ENVIRONHENTS 0F DENITRIFECATION
EN :3:
MEREEKEB LANEFSCAPE WATER
RENGVATION SYSTEM

Them: for “10 Degree of M. 5.
HICHIGAN STAYE UNIVERSETY
Loren iamea Moshier
i974

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Michigan State
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ABSTRACT

ENVIRONMENTS OF DENITRIFICATION
IN A '
BARRIERED LANDSCAPE WATER RENOVATION SYSTEM

BY

Loren James Moshier

Existence of environments of denitrification in a
Barriered Landscape water Renovation System (BLWRS) was
verified by applying a solution of nitrate as Ca(NO3)2 and
chloride as CaClz onto the BLWRS surface. Swine wastes had
been applied for extensive periods of time before experi-
mental solution application and continued to be applied
afterwards to maintain, as closely as possible, natural
conditions. Ratios of the concentration of nitrate-
nitrogen to concentration of chloride was monitored as the
solution front moved through the BLWRS soil profile.
Denitrification was presumed to have occurred in a specific
environment if reduction of nitrate-N/chloride ratio
occurred as the solution passed through that environment.
Environments in the BLWRS which were found to be conducive
to denitrification were the surface horizon and the
saturated zone. Additions of organic carbon as cracked
corn or molasses stimulated denitrification in the

saturated zone. Denitrification was also found to have

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an

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saturated zone but to a lesser degree.

ENVIRONMENTS OF DENITRIFICATION
IN A

BARRIERED LANDSCAPE WATER RENOVATION SYSTEM

BY

Loren James Moshier

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Sciences

1974

To My Wife

ACKNOWLEDGMENT S

The author wishes to gratefully acknowledge his
sincere appreciation to Dr. A. E. Erickson for his
guidance and patience at all times during this study and
in the thesis preparation.

Gratitude is expressed to Dr. B. G. Ellis, Dr. E. H.
Kidder, Dr. J. M. Tiedje and Dr. A. R. WOlcott who served
on my guidance committee.

Appreciation is extended to Mrs. Elizabeth Shields for
her assistance in laboratory analysis and to my wife,
Deanna, for typing the manuscript.

Appreciation is also extended to the Agricultural

Experiment Station for financial assistance.

TABLE OF CONTENTS

Page

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . 4
Nitrogen Cycle 4

Role of Denitrification 7
Biochemistry of Denitrification 9
5

Ecological Studies on Denitrification 1

MATERIALS MD METHODS O O O O O O O O C O O O O O O O O 2 2

BLWRS Description and Operation 22
Application of Experimental Solution 26
Sampling of Wastes, Soils, and Water 27
Methods of Analysis 29

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . 34

Waste Characteristics 35

Movement of Experimental Solution 36

Status of BLWRS Before Experiment 39

Evaluation of Amount and Environments of 43
Denitrification

CONCLUSIONS 0 O C O O O O O O O O O O O O O O O O O O O S 5

LITERATURE CITED 0 O O O O O O O O O O O O O O O O O O O 57

Table

1.

LIST OF TABLES
Page

Concentrations of nitrogen and chloride
in swine waste applied before
application of experimental
solutions . . . . . . . . . . . . . . . . . 35

Chloride concentrations in soil samples
taken from north replication site
on North BLWRS after experimental
solution had been applied . . . . . . . . . 37

Nitrate-nitrogen concentrations in soil
samples taken from north replica-
tion site on North BLWRS after
experimental solution had been
applied . . . . . . . . . . . . . . . . . . 38

Chloride concentrations in mg/liter in
water samples taken from the
saturated zone from south replica—
tion site on North BLWRS after
experimental solution had been
applied . . . . . . . . . . . . . . . . . . 39

Nitrate-N/chloride ratios in soil and
and water samples taken from.North
Swine BLWRS before application of
experimental solution. System had
undergone a 7—day aeration period
before sampling . . . . . . . . . . . . . . 40

Nitrate-N/chloride ratios in soil and
water samples taken from South
Swine BLWRS before application of
experimental solution. System had
undergone a 7-day aeration period
followed by a 5-day waste applica-
tion period before sampling . . . . . . . . 40

Concentrations of Kjeldahl-nitrogen
expressed in‘Ag/gram in soil
samples taken from North and South
Swine BLWRS before application of
-experimental solutions . . . . . . . . . . 42

Table Page

8. Moisture content (dry weight basis) of
initial soil samples from North
and South Swine BLWRS respectively . . . . 42

9. Nitrate-N/Cl ratios determined from
analysis of soil and water samples
taken from.North Swine BLWRS -
north replication site after appli-
cation of experimental solution.
System had undergone a 7-day
aeration period before application
of experimental solution . . . . . . . . . 44

10. Nitrate-N/Cl ratios determined from
analysis of soil and water samples
taken from.North Swine BLWRS -
south replication site after
application of experimental solution.
System had undergone a 7-day aeration
period before application of
experimental solution. . . . . . . . . . . 45

11. Nitrate-N/Cl ratios determined from
analysis of soil and.water samples
from South Swine BLWRS - north
replication site after application
of experimental solution. System
had undergone a 7-day aeration period
followed by a 5-day waste application
before application of experimental
solution . . . . . . . . . . . . . . . . . 46

12. Nitrate-N/Cl ratios determined from
analysis of soil and water samples
taken from South Swine BLWRS -
south replication site after
application of experimental solution.
System had undergone a 7-day aeration
period followed by a 5-day waste
application before application of
experimental solution. . . . . . . . . . . 47

13. Selected ratios of experiment solution
and per cent changes between these
ratios as solution passes through
various locations in the North and
South BLWRS. . . . . . . . . . . . . . . . 48

Table Page

14. Oxidation-reduction potential measurements
made in the Swine South BLWRS during
the Summer of 1972 in millivolts . . . . . 49

15. Numbers of anaerobes and denitrifiers
found in North Swine BLWRS soil
collected in November 1972 . . . . . . . . 53

Figure

1.

LIST OF FIGURES

Nitrogen flows in a land waste disposal
system O O O O O O O O O O O O O 0

Schematic diagram summarizing the
electron transport as found in
several denitrifying bacteria . .

Schematic diagram of BLWRS and
accompanying soil reactions. . . .

Drawings for the BLWRS showing dimen-
sions of polyvinyl barrier . . . .

Location of energy sources, effluent
catchments and wellpoints in
Swine BLWRS. . . . . . . . . . . .

Page

13

17

23

25

INTRODUCT ION

Renovation of wastewaters from agricultural and
municipal sources is a serious problem that faces present
day society. The trend toward geographical concentration
of human and domesticated animal populations has created
and magnified the problem. Plant products are grown on
large land acreages, transported for considerable distances
in most cases and consumed by large populations concen-
trated on small land areas. In the past, waste by products
were usually absorbed and renovated by large volumes of
soil and water resources located at or near locations of
plant nutrient removal. In recent years, however, a
considerable fraction of our water resources have become
polluted due to discharge of wastes in the forms of
treatment plant effluents, sewer tile effluent, runoff and
seepage from feedlot manures. The failure to return
nutrients present in these wastes to the soil and pollution
of water resources have disrupted the ecosystems of both
of these environments. The result has been eutrophication
of surface waters, aquifer contamination, and an increase
in dependency on limited fertilizer resources.

Irrigation of raw and treated wastes on well managed
soils has been presented as a solution to the waste

renovation problem. The soil can be used as a physical,

l

chemical and biological filter in this renovative process.
Nitrogen, the nutrient of concern in this thesis, is
removed from applied waste waters by the combined action
of all three components of the soil filter. The soil
surface acts as a physical filter by sieving out nitrogen
containing suspended particulate solids present in wastes
(Erickson 35 El, 1971; Thomas, 1973). Chemically, the
soil "filters out" organic nitrogen compounds and ammonium
cations via absorption on particle surfaces (Ellis, 1973).
The biological component of the soil filter, consisting of
both plants and soil microorganisms, then transforms the
various nitrogen compounds that have entered the soil
environment into various innocuous forms (Miller, 1973).
Nitrate, a potential pollutant, which enters the soil
solution directly as a wastewater constituent or as a
product of various soil microbial processes can be

removed by this "living filter”. Thus, applications on
land of agricultural and municipal wastewater can be used
to reclaim and fertilize land and to return water in a
purified state to underground supplies.

This investigation was designed to examine the role
that denitrification, the microbial transformation of
nitrate to nitrogen gas, plays in a soil system designed
to renovate water carrying suspended animal waste. The
objectives of this study were: 1) to determine the amount

of denitrification, 2) to determine the locations of

denitrification in this system, and 3) to determine the

environmental conditions of these locations.

LITERATURE REVIEW

Nitrogen Cycle

 

Several authors (Campbell and Lees, 1967; Delwiche,
1970; Jansson, 1958; Stevenson, 1965; Wblcott, 1972) have
described the nitrogen cycle in the biosphere. A flow
diagram, presented in a recent study (Wblcott, 1972)
includes the introduction of treated and untreated wastes
with low C/N ratios into the nitrogen cycle. This
diagram (Figure 1) illustrates the importance of plant
uptake and microbial transformations in the removal of
nitrogen from wastewaters. Plant uptake of nitrate
(N03) referred to as plant assimilatory nitrate reduction
(Arrow 1, Figure 1) can be significant if crops such as
corn or reed canary grass are grown and harvested on
waste disposal sites (Sopper and Kardos, 1973). The
microbial reactions involved in nitrogen removal are
mineralization, immobilization, nitrification and
denitrification.

Mineralization is the microbial conversion of
organic nitrogen to ammonium-nitrogen (Arrow 2). Immobil-
ization (Arrow 3) which includes both ammonium assimila-
tion and assimilatory nitrate reduction is the microbial

conversion of inorganic nitrogen to organic nitrogen.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure l. Nitrogen flows in a land waste disposal system.
=: Primary flow
-—%> Intermediate flow, or net flow
--4>.Restricted flow, or intermittent flow

(Adapted from Wolcott, 1972)

Mineralization and immobilization are essentially reverse
reactions with the net flow of nitrogen being dependent
on the ratio of carbon to nitrogen present in the soil
environment. If the C/N ratio of wastewaters being
applied is less than or equal to 10, the average C/N ratio
in living cells, then net mineralization will occur in the
soil (Miller, 1973). C/N ratios in swine wastes, which
were used in this investigation, were calculated using
biological oxygen demand (BOD) values and total nitrogen
values reported in the literature. Concentrations of
carbon present in the wastes were estimated by using the

, . 12 (a.w.-C) _
fOllOW1ng formula. BOD x 32 Im.w.-02) x 0.75 - C. The

 

use of this equation assumes that carbon in the presence
of oxygen is transformed to C02 and that oxidation of fatty
acids and inorganic nitrogen are accounted for by the
factor 0.75 (Wblcott, 1974). The calculated C/N ratios
were 0.33 (Erickson g; a1, 1974), 0.95 (O'Callaghan, Dodd
and Pollock, 1973), 1.43 (Ngoddy at al, 1971), 1.75 (Hart
and Turner, 1965), and 1.89 (Taiganides at al, 1964).
This low energy/N ratios in wastes must be considered when
flows of nitrogen in the soil environment are outlined
(Wblcott, 1972) as shown in Figure 1.

Nitrification (Arrows 4 and 5) is the microbial
oxidation of ammonium to nitrate by the combined action of

the chemoautotrophs Nitrosomonas and Nitrobacter.

 

 

Denitrification, sometimes referred to as dissimilatory

nitrate reduction (Arrows 6, 7 and 8), is the microbial

reduction of nitrate to nitrous oxide or nitrogen gas. In
summary, nitrate enters the soil solution as a wastewater
constituent or as a product of the mineralization-
nitrification reaction sequence and is removed by assimila-
tory nitrate reduction by plants and microorganisms and by
denitrification. Nitrate not removed from the percolation
stream by immobilization or denitrification during passage
through soil environments influenced significantly by

plant roots or microorganisms is transported to the aquifer
in the leachate (Arrow 9).

Summaries and reviews have been published on the
various processes occurring within the nitrogen cycle.
Processes which have been studied in detail are:
nitrification (Alexander, 1965), microbial assimilatory
nitrate reduction (Payne, 1973), plant uptake of nitrate
and ammonium (Viets and Hageman, 1971), nitrogen fixation
(Postgate, 1971), humification (Schnitzer and Khan, 1972)
and nitrate leaching (Viets and Hageman, 1971). In
addition, the thermodynamics and kinetics of energy trans-
fer which closely parallel cycles Of carbon and nitrogen in
natural systems have been extensively formalized (Bolin,

1970; Delwiche, 1967, 1970).

Role of Denitrification

 

 

The presence of nitrate in the aquifer is a pollution
hazard to both humans and animals. Federal health

standards (U. S. Department Health, Education, and welfare,

1962) call for less than 10 ppm nitrate-nitrogen in water
used for human consumption. Nitrate upon reduction yields
nitrite (NOE) which is commonly regarded as ten times as
toxic as nitrate when present in drinking water, food and
feeds (Viets and Hageman, 1971). Nitrite has been shown to
have deleterious effects on red blood cells (Gruener and

+2)

Shuval, 1970). Nitrite can overoxidize the ferrous (Fe
iron of hemoglobin to ferric (Fe+3) iron. The resulting
methehemoglobin in red blood cells is incapable of carrying
oxygen. Nitrate can also combine irreversibly with hemo-
globin to form nitrohemoglobin which like carbon monoxy-
hemoglobin cannot carry oxygen. Nitrosoamines formed by
reaction of nitrite with secondary amines in an acidic
environment are carcinogenic, teratogenic (tendency to
produce monstrous growths or fetuses) or mutagenic
(Liiinsky and Epstein, 1970). Nitrite can also combine
with myosin, a filamentous protein component of skeletal
muscle (Gruener and Shuval, 1970).

Aquatic forms of life are also endangered when nitrate
enters a lake or stream via seepage of polluted underground
water. The process of enrichment of aquatic systems with
nutrients, especially nitrogen and phosphorous, has been
termed eutrophication. Undesirable features of eutrophica-
tion are a change in the type of algae to a blue-green
algae form which produces obnoxious blooms and an excessive
growth of macrophytes. This change in lake flora upsets

the base of the normal food chain and results in the

creation of new food chains that lead to far less desirable
species of fish (Provasoli, 1970). Provasoli (1970) also
states that algae can accumulate in eutrophic lakes because
of reduced grazing by the aquatic herbivores. As a result,
large quantities of organic matter are produced in eutro-
phic lakes. Rapid degradation of this Organic matter re-
sults in depletion of the lake's dissolved oxygen supply.
Depletion of oxygen then results in the death of fish and
other desirable forms of aquatic life.

Presence of nitrate in soil systems designed to
renovate wastewaters is a dangerous pollutant unless it is
removed in a harmless manner. Denitrification plays this
very important role in renovation of wastewaters applied on

soils.

Biochemistryg£_Denitrification

 

 

Denitrification can be defined biochemically as a
series of anaerobic respiratory processes which reduce
nitrate to elemental nitrogen. Various facultatively
anaerobic organisms called denitrifiers utilize denitrifi-
cation as an energy yielding type of respiration to
generate adenosine triphosphate (Payne, 1973). Mixed
cultures of denitrifying microbial populations found in
sewage sludge have been shown to make use of glycolysis,
the pentose phosphate shunt and the tricarboxylic acid
cycle (DuToit, Toerien, and Davies, 1970). Genera of
bacteria, reported to be capable of denitrification

include: Achromobacter, Alcaligenes, Bacillus,

 

10

Chromobacterium, Corynebacterium, Halobacterium,

 

 

Hypomicrobium, Micrococcus, Moraxella, Nitrosomonas,

 

 

 

 

Propionibacterium, Pseudomonas, Spirillum, Thiobacillus,

 

 

 

 

and Xanthromonas (Payne, 1973). In addition, a compilation

 

of thirty three other genera of bacteria known to be
nitrate respiring (NOE———€>NO§) has been done although

some of these may actually be denitrifiers in which further
reduction has not been recognized (Payne, 1973).

Denitrifiers can utilize either oxygen or nitrate as
an electron acceptor and therefore are called facultative
anaerobic heterotrophs. The mean energy yield for the
transfer of a molar equivalent of electrons from an organic
compound to oxygen has been determined as 26.5 Kilocalories
(Payne, 1970) whereas the energy yield of an identical
transfer of electrons to nitrate is approximately 18
Kilocalories (McCarty, 1972). It is obvious then that
denitrifying organisms will be utilizing aerobic respira-
tion whenever possible.

In recent years much information has been obtained
through research on the effects of aerobiosis on denitrify-
ing organisms. An experiment was conducted (Collins, 1955)
which revealed that even in presumably aerated cultures,

the ability of Pseudomonas aeruginosa to gain the capacity

 

for nitrate respiration was directly related to the shape
of the culture flask employed. Growth in shaken flasks
with gradually more confining structures yielded cells that

approached the control (cells respiring anaerobically) in

11

nitrate-reducing ability. Oxygen was found to inhibit the
onset of synthesis of the nitrate reductase complex used

for denitrification by Enterobacter aerogenes (Pichinoty)

 

 

and D'Ornano, 1961). Production of nitrate reductase in

cultures of Bacillus stearothermophilus is inversely re-

 

lated to the quantity of oxygen present (Downey, 1966).

Studies done on Escherichia coli indicate that nitrate

 

reductase, once formed, is repressed by oxygen functioning
as an electron acceptor rather than oxygen per se (Simoni
and Shullenberger, 1972). Anoxia has been found to
derepress biosynthesis of nitrate reductase in several
bacteria (Showe and DeMoss, 1968; DeGroot and Stouthamer,
1970).

Indications are that a variance exists among denitri-
fiers in their response to oxygen levels in their immediate

environment. Studies conducted with Spirillum, Pseudomonas

 

and Xanthomonas showed that some require anoxia for

 

initiation and continuation of denitrification whereas
other organisms were denitrifying at gaseous phase oxygen
tensions as high as 153 mm (VOn Mechsner and Wahrmann, 1963).

In cultures of Haemophilus parainfluenzae, nitrate reductase

 

synthesis occurs when the quantity of oxygen is lowered to
approximately lOOyIm (Sinclair and White, 1970).

The effect of nitrate concentrations on nitrate
reductase synthesis and activity has been found to vary
among denitrifying organisms. Production of nitrate re-

ductase in Bacillus stearothermophilus is directly related

 

12

to the concentration of nitrate (Downey, 1966). Several
denitrifying soil isolates show optimal growth when the
nitrate concentrations in the media are 0.1% to 0.5%
(Bollag, Orcutt and Bollag, 1970). On the other hand,

Haemophilus parainfluenzae, Bacillus licheniformis and some

 

 

species of Enteriobacteriaceae are able to synthesize

 

nitrate reductase in either the presence or absence of
nitrate (Sinclair and White, 1970; Schulp and Stouthamer,
1970). The conflicting observations may be resolved by
considering the nitrogen status of the organism undergoing
change in environmental aeration status. Nitrogen depleted

Bacillus stearothermophilus cells synthesize nitrate

 

reductase more rapidly when transferred to anaerobic
nitrate-containing media than do fully nourished cells that
are grown aerobically before transfer (Downey and Nuner,
1967).

Because denitrification is a respiratory process, an
organic substrate is required as an electron donor. An
array of carbohydrates, alcohols, organic acids, or organic
components of complex culture media can support denitri-

fiers. The rapidly growing Mycobacterium species are able

 

to utilize seventeen different carbohydrates (Bonicke and
Kazda, 1970). Methanol is known to support growth of a

gypomicrobium species (Sperl and Hoare, 1970). Short

 

chained fatty acids and related organic acids increase

nitrate reductase activity of Mycobacterium tubercolosis

 

and lactic acid is used as an electron donor in

l3

Mycobacterium scrofulaceum species (Bonicke, Juhasc and

 

Diener, 1970). Enumeration of denitrifiers is usually
accomplished on Difco nitrate broth containing a beef
extract, in addition to supplementary amino acids, and
potassium nitrate as a growth medium (Focht and Joseph,
1973). The metabolism of the well known chemolithotrophic

bacterium Thiobacillus denitrificans is unique since an

 

oxidizable inorganic sulfur compound is used as electron
donor.

Initial events in electron transport which result in
dissimilatory nitrate reduction occur via the use of pyri-
dine nucleotides, flavines, and quinones in a manner
similar to those which occur in aerobic respiration. Trans-
port of electrons beyond the quinone stage then occurs via
a branch at the cytochrome b level (Payne, 1973). A sche-
matic diagram summarizing the electron transport system as
found in several denitrifying bacteria is presented in

Figure 2.

organic substrate

Formate
A second c-type
b- type > cytochrome
cytochrome

T 3+ 2 -

NAD—e-Flavines—e»Quinones—a-cyto bI—*[Mo(V)'Fe -S -]—4>NO3

Figure 2. Schematic diagram summarizing the electron trans-
port as found in several denitrifying bacteria
(Payne, 1973).

14

Studies have shown that an increase in c-type cyto-
chrome synthesis occurs accompanied by a decrease in a-type
cytochromes when several organisms begin reducing nitrate
(Downey and Kiszkiss, 1969; Schulp and Stouthamer, 1970;
Sinclair and White, 1970). Membrane-associated and soluble
cytochromes have been found to assist in transfer of
electrons beyond nitrate in the following manner:
NOS——e> NOE—34>NO —e—>N20 —e—>N2 (Cox, Payne and
Dervartian, 1971). These cytochromes may be considered as
cofactors of the various reducing enzymes which include the
nitrate, nitrite, nitric oxide and nitrous oxide reductases.

Because a complex of enzymes are involved in transport
of electrons from one nitrogenous oxide to the next, roles
of N2 production are dependent on temperature and pH of the
surrounding environment. Various optimum temperatures for
N2 production have been reported, depending on the species
involved. A psychrophilic pseudomonad_has been found with
an optimum temperature less than 20°C (Konishi, 1969) while
certain thermophilic denitrifiers show optimum temperatures
as high as 55°C (Downey and Kiszkiss, 1969). Incubation
temperatures near 30°C were found to favor denitrification
by several soil isolates (Bollag, 33 a1, 1970). In this
same study, pH values slightly above neutrality were found
to favor denitrification.

In summary, the biochemical requirements for denitri-
fication are: l) denitrifying populations, 2) anaerobic

conditions, 3) nitrate as an electron acceptor, 4) readily

15

available source of carbonaceous compounds as electron
donors, 5) moderate temperatures, and 6) a slightly

alkaline pH.

Ecological Studies on Denitrification

 

 

As discussed previously, anaerobic conditions are
essential if denitrification is to occur. Anaerobiosis
exists in the field when rate of oxygen utilization exceeds
rate of diffusion to a particular site or when rate of
diffusion is insignificant to a particular site. Anaerobic
sites are possible whenever respiratory consumption of
oxygen by plant roots and microorganisms is taking place
(Stefanson, 1972; WOldendorp, Dilz and Kolenbrander, 1966)
and include microenvironments, macropores or entire layers
within aerated soils (Broadbent, 1973). The presence of
water in an environment also allows that environment to
become anaerobic since oxygen which has been displaced
diffuses very slowly in water. Anaerobic microenvironments
may exist where films of water surround soil particles.

The presence of anaerobic microenvironments allows plant
uptake, organic material decomposition, nitrification and
denitrification to occur in the same soil macroenvironment.
This allows renovation of wastewaters containing various
forms of nitrogen to occur when applied to an aerated soil.
Saturated soils represent anaerobic macroenvironments.
These include soils above the water table if rates of water

application are very high, and soils at or below the water

16

table. Redox potentials equal to or less than a positive
200 milli-volts indicates that an environment is sufficient-
ly anaerobic for denitrification to occur (Miller, 1973).

During the past several years, an extensive study has
been conducted concerning renovation of water from livestock
liquid wastes by modified soil system (Erickson gt_al, 1971;
1972; 1974). This modified soil system, called a Barriered
Landscape Water Renovation System (BLWRS) was designed to
remove nitrogen as well as phosphate and carbonaceous
materials present in animal wastes by allowing various soil
reactions to occur. The soil reactions important in this
renovative process are mineralization (decomposition of
organic matter), nitrification, denitrification and phos-
phate fixation. A schematic diagram Of the BLWRS with
accompanying soil reactions is shown in Figure 3.

Nitrate not denitrified in anaerobic microenvironments
in the aerobic zone enters the saturated soil (anaerobic)
zone created by installation of a water barrier. As the
nitrate is forced to move laterally through the anaerobic
soil, it is denitrified to nitrogen which diffuses to the
soil surface. In a pilot system constructed in May, 1970,
and Operated during the following summer months, 99.5 per
cent of the nitrogen was removed from the original applied
swine waste (Erickson g; a1, 1971). Swine and dairy liquid
wastes were applied for 17 months and 15 months respective-

ly on larger BLWRS constructed in spring, 1971. In these

17

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18

studies, 80 per cent and 97 per cent respectively of the
nitrogen was removed (Erickson gt 21, 1974).

In the BLWRS, intermittent applications of wastes was
found to be effective in promoting the nitrification-
denitrification sequence. Other investigators have also
determined that intermittent applications of water on soils
results in considerable nitrogen removal (Bouwer, 1973;
Broadbent, 1973; International Rice Research Institute, 1969;
Viets and Hageman, 1971). In the Flushing Meadows Project
(Bouwer, 1973) a series of basins were flooded intermittent-
ly with municipal wastewater which was allowed to percolate
through underlying soil layers. During flooding an anaero-
bic environment was formed which allowed nitrate to be
reduced to N2 by denitrification. At the same time, nitrogen
in the ammonium form was being adsorbed by the clay and
organic matter. During the following dry-up period the
ammonium was then nitrified to nitrate and became subject
to denitrification when the next flooding occurred. Thirty
per cent of the nitrogen applied was estimated to have been
removed. In a laboratory study conducted on an intermittent-
flooded column of Tulare sandy loam, 83 per cent of the
nitrogen added was removed and presumed to be denitrified
(Broadbent, 1973).

Nitrate applied as fertilizer has been found to
disappear as the soil solution approaches the water table
(Kolenbrander, 1972; Meek, Grass and MacKensie, 1969). In

one of these studies (Meek §E_al, 1969) nitrate

19

concentrations and redox potential values were measured as

a function of depth in a cotton field of high water table

in the Imperial Valley, California, after application of 280
kilograms anhydrous ammonia per hectare. Reduction in
nitrate concentrations and a drop in redox potential were
found as the soil solution approached the water table.

An experiment conducted on one-meter columns of Venice peat
revealed that a striking contrast exists in amount of
denitrification occurring in a partly saturated soil com-
pared to an unsaturated soil (Broadbent, 1973). Venice
peat is a very permeable soil and contains forty per cent
organic carbon. One column was free draining; the other had
the water table maintained at 70 cm from the surface.
Recovery of tagged nitrate in effluents Obtained at the
bottom of the column revealed that 21 per cent of the added
nitrate was recovered from the unsaturated column whereas
less than one per cent of the added nitrate was recovered
from the column with the 30 cm saturated layer.

The importance of a readily available source of organic
compounds has been realized in systems designed to renovate
nitrate-contaminated waters. Sucrose, lactose and methanol
have been employed as electron donors in purification of
waters (Klotter, 1970; McCarty, Beck, and St. Amant, 1969).
Use of methanol has proven feasible since methanol when
metabolized, yields nitrogen gas, carbon dioxide, water,
and a small crop of cells (McCarty gt_§1, 1969). A 2:1 to

3:1 ratio of methanol to nitrate is indicated by model

20

experiments since greater ratios may leave residues that
contribute to unwanted increases in biological oxygen
demand (BOD) of treated waters (Dholakia, Stone and Burch-
field, 1970).

In soil systems, there is evidence that vegetation has
a beneficial effect on denitrification (Bouwer, 1973;
Broadbent, 1973; Wbldendorp 23 31, 1966). As already
mentioned, plant roots consume oxygen and therefore create
anaerobic pockets within the rhizosphere. However, the
vegetation also may have a beneficial effect on denitrifi-
cation by providing energy to the denitrifiers in the form
of organic carbon exuded by live roots and that returned to
the soil by decaying plant debris (WOldendorp gt 31, 1966).
In one study (WOldendorp §E_gl, 1966) it was noted that
fifteen to twenty per cent of the nitrate passing through
the rhizosphere may be denitrified.

When wastewaters are applied to vegetated areas,
removal of additional nitrate occurs due to incorporation
into microbial cells supported by organic carbon present in
wastes and in vegetative exudates and detritus (Miller,
1973). To ensure greater efficiency of nitrate removal in
soils, however, it may be necessary to add oxidizable
organic materials into the soil environment, especially at
lower depths. Saturated soil environments containing
molasses or corn were found to be more effective in removing
nitrate when compared to the soil environment containing no

additional energy source (Erickson et_al, 1974). The

21

experiment conducted on the partly saturated Venice peat
soil containing 40 per cent organic carbon (Broadbent,
1973), mentioned earlier, revealed that essentially all
nitrate can be removed from the leachate when sufficient
organic substrate is available.

Soil temperature also has been found to be an important
parameter in waste disposal on land. In the BLWRS, tempera-
ture of the soil environment was found to effect the
concentration of nitrate in the effluents (Erickson gt a1,
1974). By November, soil temperatures in the anaerdbic
zone reached 10°C or lower and at the same time the concen-
tration of nitrate in the effluents increased greatly.
Reduced efficiency of denitrifiers at this temperature or

lower seemed to be responsible.

MATERIALS AND METHODS

BLWRS Description and Operation

 

 

The Barriered Landscape Water Renovation System (BLWRS),
a modified soil system designed to renovate animal wastes,
was used in this investigation.

A pair of BLWRS, referred to as North and South Swine
BLWRS, were constructed of fine sand in the Spring of 1971
at the Swine Research Facility at Michigan State University.
Each BLWRS was 10.7 meters wide and 15.25 meters long with
an anaerobic zone (saturated zone) 30.5 cm deep in the
middle and 61 cm deep in the ends overlain by an aerobic
zone (unsaturated zone) 1.5 meters deep within the waste
application area. A uniform slope began 3.8 meters from
each end and continued down to the end of the BLWRS. These
slopes were designed to promote better aeration within the
upper layers of the BLWRS. The dimensions of the BLWRS are
illustrated in a three-view diagram (Figure 4).

The barrier was made of 30 mil polyvinyl sheeting which
was turned up at the edges and sealed in the corners to form
a complete basin. The only outlet was the tile opening
which was reduced to 1.25 cm, the inner diameter of tygon
tubing which was inserted into a tile plug. This tygon

tubing loop was approximately one meter in length and was

22

Figure 4.

23

 

 

 

 

 

 

 

 

 

15.25m

I r_n.

1 I '
I l l
I I I
: . .
I : I
: I10.7m '
. : '
I I I
I . I
I I I
I . '
' __!J.

Top View End View

 

 

 

3.8m
F f Ebro—.9!“ 2.1m

Side View

 

 

 

Drawings for the BLWRS, showing dimensions of
polyvinyl barrier.

24

raised to maintain the level of the saturated zone at 30.5
cm from the bottom of the BLWRS.

The Swine BLWRS, which had been in operation from the
Summer of 1971 until the Fall of 1972 was again put into
operation in June 1973 in order to conduct a study on
denitrification. Swine liquid wastes collected from a
slotted floor, flush type, eighty-sow gestation barn were
automatically sprayed on the BLWRS surface with an automatic
boom spray apparatus which passed over the BLWRS every 30
minutes. Each BLWRS was sprayed for seven days and then
allowed to dry out for seven days; this type of intermittent
spraying was used in order to promote the nitrification-
denitrification reaction sequence and to promote decomposi-
tion of slimes which had been filtered out on the surface.

A vegetative cover (quack grass, rye grass and various
weeds) was maintained in order to increase infiltration and
to provide an environment (rhizosphere) conducive to denitri-
fiers. Also energy in several forms was provided for
denitrifiers. In the Summer of 1971, a trench was dug on
the west end of the North Swine BLWRS; this trench was filled
with approximately 1000 pounds of cracked corn. During
construction, a perforated pipe 1.9 cm in diameter was laid
along the edges and bottom of the east end of each BLWRS;
in July 1973 these pipes were each filled with three liters
of molasses. No energy was added to the west end of the
South Swine BLWRS; this end served as the control. The

location of the energy zones can be seen in Figure 5.

25

 

OW Ow W0
D C North M D
BLWRS
()W o W WO

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(3w C>w . wt)
D South M D N
BLWRS
ow 0w wo
Top View
“ 15.25m I
4—3.8
l
‘ l
W C W M
D Side View D
M = molasses energy source
C = corn energy source
E = effluent catchment
D = drain
W = wellpoint

Figure 5. Location of energy sources, effluent catchments
and wellpoints in Swine BLWRS.

26

Swine waste was applied on the Swine BLWRS intermittent-
ly as previously described for a period of two months.
During this period, it was felt that a type of equilibrium
would be established among the various soil reactions
occurring in each Swine BLWRS. During this period, 44,034
liters in 30 days and 53,025 liters of waste in 40 days were
applied on the South and North Swine BLWRS respectively.
Averages of waste applied were 1,468 liters and 1,326

liters of 1.80 cm and 1.63 cm per day respectively.

Application gf_Experimental Solution

 

On August 14, 1973, 78.5 liters of a solution contain-
ing 10.19% nitrogen as Ca(N03)2 and 4.57% chloride as CaCl2
was applied on the South Swine BLWRS. This sytem had
undergone a seven day aeration period followed by a five day
waste application period before the solution was applied.
The solution was made up in tap water and sprayed onto the
soil surface in the same manner that waste was applied. On
August 30, 1973, 82.3 liters of a solution containing 12.24%
nitrogen and 4.99% chloride was applied on the North Swine
BLWRS. This system had just undergone a seven day aeration
period before the solution was applied. Swine wastes were
applied intermittently on the Swine BLWRS after application
of the solutions until the experiment was terminated in
November.

A concentrated chloride solution was used in order that

a chloride peak could be followed down through the soil

27

profile of the BLWRS. Chloride was used as a tracer since
it is a mobile anion in soils and undergoes essentially no
microbial transformations. A tracer was required so that
dilution of the experimental solution in the soil environ-
ment would be accounted for. Nitrate, an anion of similar
mobility, would be used as a microbial substrate (electron
donor) in the denitrification process. Ratios of concentra-
tion of nitrate-nitrogen to concentration chloride in the
samples containing the chloride peak were then calculated
and changes in these ratios Observed to determine the
extent to which nitrate was being used as a substrate by
denitrifiers. In further discussions, these ratios shall

be referred to as nitrate-N/chloride ratios.

Sampling of wastes, Soils, and Water

 

 

Waste samples were collected in narrow mouth 250 m1
polyethylene bottles periodically throughout July from a
sampling port which had been installed into the plumbing
system of the spray boom apparatus. A total nitrogen and
chloride analysis was made on these samples in order that
a nitrogen/chloride ratio could be determined. The experi-
mental solutions were made up such that the ratio of nitro-
gen to chloride was similar to that present in the wastes.

The soil profile of each BLWRS was sampled.within the
spray area just prior to application of experimental
solutions and then at intervals until the chloride peak had

moved into the saturated zone. Both north and south sides

28

of each BLWRS were sampled; therefore the experiment was
conducted in duplicate. Soil samples were taken at the
following depths: 0-5, 5-10, 10-15, 15-30.5, 30.5-46,
46-61, 61-76, 76-91.5, 91.5-107, 107-122, 122-137, and
137-152 cm. A soil sampling probe (cutting edge of 1.9 cm
diameter) was used to take samples from 0 to 30.5 cm.
Three to six different soil samples were taken and then
mixed and placed into sealed moisture proof plastic bags.
Samples in the remaining portion of the profile were taken
with a soil auger (2.5 cm diameter) in duplicate, mixed and
placed in similar bags.

Well points were placed in the BLWRS in order that
water samples could be taken from the saturated zone. These
well points were made from thin-walled aluminum tubing 2.5
cm in diameter which were perforated throughout a 25 cm
portion on the end. The well points with plastic plugs
inserted in the ends were placed in the BLWRS in holes pre-
viously dug with the soil auger. Two well points were
placed in the center within the spray area of each BLWRS and
two well points were placed on each end. Figure 5 shows the
location of the various well points. When the ends of the
tubes were 25 cm below the interface between the saturated
and unsaturated zone, the plastic plugs were then removed.

Water samples were taken from the well points by an
evacuation apparatus consisting of a vacuum tank, an erlen-
meyer flask as a trap, and vacuum hose. The first sample

was discarded due to the higher aeration status within the

29

well point compared to the soil environment. The well
points were allowed to recharge and then water samples were
collected in narrow mouth 250 ml polyethylene bottles.

Two samples had to be taken from each well point since the
volume of the tubing within the saturated zone was approxi-
mately 125 ml.

Water samples were taken from the saturated zone after
waste had been applied for several days and continued to be
taken until the chloride peak appeared in the tile effluent.
water samples were also taken from the tile outlet. Figure
5 shows the locations of the tile outlets in the North and
South Swine BLWRS respectively.

All wastes, soil and.water samples were stored at 4°C
in a walk-in cooler and were analyzed as soon as possible
for chloride, nitrate, and in some cases other forms of

nitrogen.
Methods of Analysis

Waste samples were analyzed for ammonium, Kjeldahl,
organic, nitrate and nitrite-nitrogen and chloride. Ammon-
ium nitrogen was analyzed by steam distillation procedure.
Two ml of waste were pipetted into a micro distillation
flask and the flask then attached to a micro steam
distillation apparatus. Ten m1 of 0.1 N NaOH were added
to the sample via a tube connecting a reservoir to the flask.
The solution was steam distilled as rapidly as possible and

the distillate was collected in a 50 ml erlenmeyer flask

30

containing 5 ml of a 2 per cent boric acid solution and 1
drop of Fleisher methyl-purple indicator.1 The ammonium
present was then titrated with H2804 solution which had
been standardized by use of THAM-tris (hydroxymethyl) amino
methane2 as the primary acidimetric standard. Concentra-
tions of ammonium-nitrogen in the samples were then
calculated.

Kjeldahl nitrogen (organic nitrogen plus ammonium nitro-
gen) was analyzed by the semi-micro Kjeldahl method (Bremner,
1965a). TWO m1 of waste were pipetted into a micro distilla-
tion flask and two m1 of concentrated H2804 were then added.
Approximately one-third gram of a mixture containing K2804,
CuSO4 and Selenium was added to the solution. Potassium
sulfate (K2804) was used to raise the boiling point of

H SO4 and thus the temperature at which digestion could

2
occur whereas CuSO4 and Se were used as catalysts to
promote oxidation of organic matter (Bremner, 1965a; Skoog
and West, 1969). The sample then was placed on micro
digestion plates and digested for several hours. Samples
were assumed to be completely digested after clearing of
H2804 fumes had occurred. After allowing the sample to

cool, 10 m1 of distilled water were added. The sample then

was steam distilled using the same procedure discussed

 

1Can be Obtained from Fleisher Chemical Company,
Washington, D. C. '

2Can be obtained from Fisher Scientific Company, Chicago,
Illinois.

31

above except that 10N NaOH was used as the alkali. Concen-
trations Of Kjeldahl nitrogen were then calculated.

Since Kjeldahl nitrogen represents organic nitrogen plus
ammonium nitrogen, organic nitrogen than can be calculated
by subtracting concentrations of ammonium nitrogen as
determined by the steam distillation procedure from Kjeldahl
nitrogen as determined by the semi-micro Kjeldahl method.

Nitrate-nitrogen was determined by an electrode proce-
dure. The apparatus used consisted of the Orion Nitrate
Specific Ion Electrode, Model 92-07; a calomel reference
electrode, and a Model 801 digital pH/mv meter. Standard
solutions containing 5, 10, 50, 100, 200 ppm nitrogen as
KNO3 in distilled water were prepared and a standard curve
Of concentration versus millivolts was made. Nitrate-
nitrogen in both wastes and standard solutions was analyzed
by introducing approximately 30 milliliters of the solution
into a 50 ml beaker, stirring the solution with a magnetic
stirrer and immersing the electrodes into the solution.
Concentrations of nitrate-nitrogen in the wastes was then
determined by use of the standard curve.

Nitrite-nitrogen in the wastes was analyzed by the
Modified Griess-Ilosvay Method (Bremner, 1965b). One-third
m1 of sample was pipetted into small plastic tubes, then 2
m1 of 1 per cent sulfanalamide solution and 2 m1 of 0.2 per
cent N-l-Napthylethylene dihydrochloride were added. The color
was then allowed to develop for 30 minutes and transmittance

measured on a Bausch and Lomb Spectronic 20 at a wavelength

32

of 540 millimicrons. Standard solutions of 0, 0.5, 1.0,
1.5, 2.0 ppm nitrogen as NaNO2 were prepared and a standard
curve then was graphed on semi-log graph paper. Concentra-
tions of nitrite-nitrogen in the wastes were calculated by
use of the standard curve.

Chloride was analyzed by a AgNO3 titration procedure.
Five ml of waste were diluted with 20 ml distilled water
and this solution then placed in a 50 m1 flask. One drop
of KOH solution was added to adjust pH from 7.0 to 10.0.
Indicator solution(0.5 m1 Of K2Cr0g was added and the
solution was titrated to a pinkish-yellow endpoint with
AgNO3 solution which had been standardized against solutions
of known NaCl concentrations. Concentrations of chloride
in the wastes then were calculated.

Kjeldahl nitrogen, nitrate-nitrogen and chloride
analysis were conducted on wet soil samples which had been
stored in moisture-proof plastic bags. Air drying of soil
samples was not done since it was feared that significant
loss of ammonium and nitrate-nitrogen could occur. Chloride
analyses were conducted on wet soils because chloride and
nitrate were analyzed in the same soil extract.

Kjeldahl nitrogen was analyzed in soil samples taken on
the day that experimental solutions were applied. TWO grams
of wet soil were placed in’a dry micro distillation flask.
Two m1 of H20 were then added and the sample was allowed to
stand for 30 minutes. Approximately 1.1 grams of K280

4

catalyst mixture and 2 m1 of concentrated HZSO4 were

33

added to the flasks which were then placed on micro digestion
plates for approximately four hours. TWenty to thirty ml of
deionized water were added after the flasks had cooled and
the samples then were steam distilled using the same
procedure as discussed for waste analysis.

Nitrate in the soil samples was analyzed by using the
electrode procedure. The same apparatus was used as
discussed in waste analysis. Twenty grams of wet soil was
weighed and placed in 125 m1 erlenmeyer flasks. Fifty m1
of saturated CaSO4 solution were added to the samples and
the samples then were shaken on a rotary shaker at 200 rpm
for 30 minutes. The nitrate in the supernatant portion of
the sample then was analyzed by electrode. Standard solu-
tions of 5, 10, 50, 100, 200 ppm nitrogen as KNO3 were made
up in saturated CaSO4 solution. A standard curve was then
developed which was used for calculation Of the nitrate
concentration of the soil samples.

Chloride in the same soil extracts also was analyzed
by an electrode procedure immediately after nitrate
analysis had been completed. The apparatus used in the
analysis consisted of Orion Chloride Specific Ion Electrode
94-17; a calomel reference electrode, and Sargent pH meter,
Model DR S-30000. Standard solutions of 1, 5, 10, 50, 100,
200 ppm C1- as KCl were used to develop a standard curve.
Concentrations then were calculated by use Of the standard

curve .

34

Nitrate and chloride in water samples taken from the
saturated zone and tile effluent were also analyzed by
electrode procedure. The water samples were allowed to
come to room temperature and nitrate-nitrogen and chloride
was analyzed directly by use of analytical equipment
previously mentioned.

Ratios of weight of wet soil sample to weight of solu-
tion in sample were also determined. weight of solution
was determined by subtracting weight of oven dried sample
from weight of wet sample. Nitrate and chloride concentra-
tions as determined by electrode analysis on wet samples
then were multiplied by these ratios in order to obtain
concentrations on a solution basis. An example of these

calculations are shown below:

 

weight Of wet soil sample = 20.00 gm

weight of O.D.S. = 18.77 gm

weight of water = 1.23 gm

weight of wet soil = 20.00 = 16 26

weight of water 1.23 '

nitrate concentration in _

wet soil sample _ 81'84 ppm

nitrate concentration in = 81.84 ppm x 16.26 = 1331 ppm

soil solution

RESULTS AND DISCUSSION

Waste Characteristics

 

Data Obtained from analysis of swine waste samples are
presented in Table 1. The average concentration of total
nitrogen was found to be 372.2 mg/liter. The ratio of
average nitrogen concentration to average chloride concen-
tration in the wastes equalled 2.36. The experimental
solutions were prepared such that nitrogen to chloride

ratios were similar to this value.

Table 1. Concentrations of nitrogen and chloride in swine
waste applied before application of experimental
solutions.

 

Date + _

of NH4-N Org N N034N NOz-N Total N C1

Samp- mg/liter mg/liter mg/liter mg/liter mg/liter mg/liter
ling

 

July 12 326.5 32.5 17.0 0.18 376.2 91.0
July 19 354.9 34.0 8.2 0.11 397.2 170.0
July 26 300.8 35.5 15.0 0.24 351.5 160.0
August 2 324.0 27.3 12.0 0.30 363.5 210.0
Average 326.6 32.3 13.1 0.21 372.1 158.0

 

35

36

Movement of Experimental Solution

 

An experimental solution containing 12.24% (122,400 ppm)
nitrogen as Ca(N03)2 and 4.99% (49,900 ppm) chloride as CaCl2
was applied on the North Swine BLWRS whereas a similar
solution was applied on the South Swine BLWRS. The use Of
chloride as a tracer proved to be successful in most cases.
The chloride peak was usually easily detected in the profile
after sampling and analysis had been completed. A direct
relationship existed between depth at which the chloride and
nitrate peak occurred and length of time waste had been
applied. Tables 2 and 3 illustrate this relationship.
Similar movements of chloride and nitrate were observed in
the other sampling sites. Movement of the chloride peak
laterally along the saturated zone also could easily be
detected and was dependent on length of time that had
passed since solution was applied (Table 4). Similar
movements of chloride in the saturated zone were observed
in the saturated zones of the other sampling sites. A
comparison between Tables 2 and 3 reveals that nitrate
concentrations usually peaked at the same location in the
profile as did chloride. This indicated that the mobility

of the nitrate anion in the soil was very similar to the

mobility of the chloride anion.

37

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me a one cofluaaom Housmafluooxm mo cowumOHHoom Hmumo mmoo mo Henson me x muons Amlxvm

 

 

 

 

H.Nsw v.mna m.mo¢ m.mmm o.~mm mmHIsma
v.HHm m.mmm m.b~ma n.man m.Hm¢ SMHINNH
H.oma ~.mov H.emm H.NOOH m.mm¢ m.NHm o.mmv NNHIsoH
m.m¢m m.¢mv m.mam h.mem H.mvHH o.hom o.mow hoHIm.Hm
m.~mm m.¢mha w.mHmH 0.0mm m.HmImh
H.~mv o.¢om m.~oha «.mam ohIHm
h.¢~h b.0wv N.Hhma H.-ma m.mav Hwva
m.m¢m m.mmm m.mom ¢.mmHH vam.om
m.aa¢ m.oam m.mmhm c.0mm m.QMImH
m.mHNH o.ma- maloa
m.mav o.m~am oaIm
«.mhm h.o¢v mIo

fiImC AAIHC SIS :33 :32 SIS SIS SIS AmIm SIC MAT:
AH 0H m m h m m v m N H caov
maofimm maofiwm OHQEMMIoHoEom Odommmoaoaom maofiom mHofimm mHoEmm mHoEmm OHmEmm gamma

mammmIcOfiusHOm Sam I meowumuuamoqmo mwwuoaoo
.omflaoom soon on: coausaom Houcmfieummxm Hmumm mmznm nunoz

so Open coflumOflHomn mono: Eonm coxmu mOHmEMO HwOm aw mooHumuucmonoo mofluoaso .m OHQOB

.coflumoeammm mummz Hmsuom mo mmmo mo Hones:
we > one coeusHOm Hmucmfiflnmmxw mo oceanowaomm kumm when no HOQESG me x mum£3 Amevm

 

38

 

 

 

m.HmmH o.mma «.mom m.ov¢ H.mm¢ NmHIsma
m.mmv m.mmm o.om¢m o.mmma ~.Ham hmHINNH
n.m>a H.mm¢ m.vH¢H m.¢hma h.mhm H.m- m.hmm NNHIBOH
¢.Hhm «.memm o.om¢a m.mn H.Hmha m.m¢m ¢.H~v SOHIm.Hm
h¢.mm v.mh¢m m.hmmw m.m~m m.HmImh
m.mmv o.am¢ H.mhmm o.qaw tham
m.mmm ~.hm~ H.mmaa m.mwvm w.mm¢ Hwva
«.mwm m.mmva m.mmwa m.mmm vam.om
H.mmH m.mmm m.oamh m.~m~ m.omImH
~.mamm m.mo¢m mHIoa
m.~¢¢ N.mmme calm
o.mmm m.oamm mIo
EImd KIHC A73 A78 SIC SIB AmImv STE SIC ANINV mSIC

Ha 0H m m e o m e m m a AEOV
maofimm maofimm OHoEmm mHoEmm maoemm mamfimm mamfimm mwmfimm maofimm OHOEmm mHmEmm Spoon

mwmmb :omusHOm 8mm I msowmmnpcmocoo mummuwz

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Open :OaumOfiHomu ounce scum smxmu mmamamm HHOm ca msowumuusmosoo ommOHuHcImumuuwz .m manna.

39

 

 

Table 4. Chloride concentrations in mg/liter in water

samples taken from saturated zone from south

replication site on North BLWRS after experi-

mental solution had been applied.
SW Tile SW Slope S Top SE Slope SE Tile
321 (54-29)a 271 (42-22) 654 (26-15) 418 (56-29) 351 (56-29)
367 (56-29) 232 (44-24) 654 (28-15) 437 (58-31) 307 (58-31)
367 (58-31) 292 (47-27) 1180 (30-17) 458 (61-34) 402 (61-34)
309 (61-34) 198 (49—29) 654 (33-20) 391 (63-36) 331 (63-36)
318 (63-36) 182 (51-29) 739 (35-22) 353 (65—36) 272 (65-36)

 

a(x-y)where x is number of days after application of experi-
mental solution and y is number of days of actual waste
application.

Status of BLWRS Before Experiment

 

Nitrate-N/chloride ratios were calculated after analysis
had been completed on the initial soil and water (tile)
samples. These data are presented in Tables 5 and 6. The
nitrate-N/chloride ratios in the North BLWRS samples are
higher than the corresponding ratios in the South BLWRS
samples. This difference is due to higher concentrations of
nitrate which were found in the North BLWRS samples. The
North BLWRS had undergone a seven day aeration period
before samples were taken whereas the South BLWRS had under-
gone a seven day aeration period which was then followed by
a five day waste application period. Wastes were continually
being applied to the South BLWRS when these samples were
taken. The improved aeration status in the North BLWRS would

have favored nitrification and increased stability of the

nitrate produced.

40

Table 5. Nitrate-N/chloride ratios in soil and water samples
taken from North Swine BLWRS before application of
experimental solution. System had undergone a 7-
day aeration period before sampling.

 

 

Depth North TOP SE Tile South Top SW Tile

Surface 0.08 0.08

0-5 cm. 3.27 5.08

5-10 1.34 2.30

10-15 0.80 1.39

15-30.5 2.52 1.41

30.5-46 0.68 1.17

46-61 0.75 0.70

61-76 0.88 0.59

76-91.5 1.10 0.84

91.5-107 1.19 0.59

107-122 1.29 0.76

122-137 1.26 0.82

137-152 0.73 1.32

Sat. Zone 0.36 0.16
Molasses Cracked
Treatment Corn

Treatment

 

Table 6. Nitrate-N/chloride ratios in soil and water samp-
les taken from South Swine BLWRS before application
of experimental solution. System had undergone a
7-day aeration period followed by a 5-day waste
application period before sampling.

 

Depth NE Tile North Top NW Tile South Top

surface 0.08 0.08
0-5 0.47 0.49
5-10 0.53 0.48
10-15 0.67 0.58
15-30.5 0.85 0.47
30.5-46 0.59 0.58
46-61 0.57 0.70
61-76 0.49 0.86
76-91.5 0.52 0.79
91.5-107 0.42 0.47
107-122 0.36 0.32
122-137 0.55 0.34
137-152 0.64 0.67
Sat. Zone 0.20 T 1.08

Molasses Control
Treatment

 

41

Nitrogen analysis by the Kjeldahl method was done on
these samples; this data is presented in Table 7. These
data shows that considerable nitrogen substrate was available
in both BLWRS for the mineralization and nitrification;
however the tendency for values to be lower in the upper
profile of the North BLWRS compared to the South BLWRS
indicates that greater net mineralization has occurred in
the former. Moisture content data of the two systems,
presented in Table 8, verifies the difference in aeration
status. Only at depths near the saturated zone did the
moisture content of the North BLWRS approach that of the
South BLWRS; therefore, more oxygen was present in the soil
profile of the North BLWRS at the time of sampling.

Decrease in aeration status as indicated by moisture
content data (Table 8), promoted denitrification in the
South BLWRS at the time of sampling. This also explains
the lower nitrate-N/chloride ratios in the South BLWRS
samples. Enough organic carbon was being added as a waste
constituent and as plant root exudates and debris to allow
some denitrification to occur. Data reported by Erickson
gt 31 (1974), indicated that an accumulation of carbon had
occurred in the soil profile of the Swine BLWRS during the
previous 17 months of operation.

Variations in nitrate-N/chloride ratios in the tile
effluent from both North and South Swine BLWRS illustrate
the importance of the presence of organic carbon at lower

depths in the soil profile. Nitrate-N/chloride ratios Of

42

Table 7. Concentrations of Kjeldahl-nitrogen expressed in
yg/gram in soil samples taken from North and South
Swine BLWRS before application of experimental
solutions.

 

 

 

NOrth BLWRS South BLWRS
Depth NortH’ South North South
(cm) Replication Replication Replication Replication
0-5 856.6 1289.6 1193.0 1622.4
5-10 .148.7 195.4 249.5 275.3
10-15 142.4 107.8 133.7 186.4
15-30.5 172.2 107.8 103.3 115.3
30.5-46 104.4 109.2 95.5 114.0
46-61 86.1 84.9 80.7 80.7
61-76 54.1 80.0 73.9 78.5
76-9l.5 55.5 46.2 131.7 80.9
91.5-107 61.2 77.2 100.6 88.4
107-122 55.0 75.1 147.9 71.1
122-137 83.3 96.4 154.3 67.0
137-152 55.2 117.8 182.8 86.3

 

 

 

Table 8. Moisture content (dry weight basis) of initial
soil samples from North and South Swine BLWRS

 

 

 

respectively.

Depth North BmRS SORT) BLWE
Lem.) North Top South Top North Top South Top
0-5 6.6% 9.7% 24.1% 33.5%
5-10 6.6 4.0 12.0 12.2
10-15 4.9 3.6 10.5 11.0
15—30.5 4.5 5.4 10.4 11.0
30.5-46 5.8 5.2 9.8 10.7
46-61 6.8 6.0 10.0 12.2
61-76 7.0 5.8 11.0 11.4
76-91.5 7.8 6.6 12.4 11.6
91.5-107 6.8 7.4 10.6 9.1
107-122 9.2 10.0 11.4 11.0
122-137 13.9 11.8 14.0 14.6
137-152 18.0 14.1 18.0 18.2

 

 

 

43

water samples are given in Tables 5 and 6. All three
organic carbon treatment sites proved to be more effective
than the control site in removing nitrate from.water passing

through these sites.

Evaluation of Amount and Environments of Denitrification

 

 

 

After the experimental solutions were applied, soil and
water samples were taken at various depths corresponding to
the location of the chloride peak in the BLWRS. The
nitrate-N/chloride ratios of these samplesane shown in
Tables 9-12. The ratio at the surface represents the
nitrate-N/chloride ratio of the experimental solution applied
to the BLWRS surface. These ratios, 2.41 and 2.32 for the
North and South Swine BLWRS experimental solutions respective-
ly,were close to the ratio Of the swine waste (2.36).

Ratios from the data in Tables 9-12 were selected
from specific locations and changes in these ratios were
evaluated. Table 13 presents this data.

Data in Table 13 shows that a 44 and 47 per cent
decrease in nitrate-N/chloride ratios occurred as the experi-
mental solution passed through the upper ten centimeters of
the South and North BLWRS profiles respectively. This is
evidence that denitrification is occurring in the surface
horizon of the BLWRS. Apparently, sufficient organic carbon
from the swine wastes and from root exudates was available
to the denitrifiers. In addition, anaerobic environments

were present even in the previously well-aerated soil to

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48

promote denitrification. Application of waStewater to the
surface would exclude oxygen from this environment; oxygen
also would be depleted because of active root respiration
and microbial decomposition of the organic matter added to

the BLWRS surface.

Table 13. Selected ratios of experimental solution and per
cent changes between these ratios as solution
passes through various locations in the North
and South BLWRS.

 

 

 

Location North BLWRSa b South BLWRSa
Ratio %Change Ratio %Change
Surface 2.41 -- 2.32 --
Center, 0-10 cm 1.27 47(-) 1.30 44(-)
Center, 10-30 cm c 2.85 124(+) 1.94 49(+)
Center, 30—152 cm 1.73 39(-) 1.77 9(-)
Sat. zone, control trtmt. —- --- 1.43 19(-)
Sat. zone, molasses trtmt. 1.48 l4(-) 0.94 47(-)
Sat. zone, corn trtmt. 0.53 6900 -——- -—
Tile, beyond control trtmt. --- —- 1.51 6(+)
Tile, beyond molasses trtmt. 1.41 5(-) 0.49 48(—)
Tile, beyond corn trtmt. 1.21 128(+) -— -—

 

 

aValues for center, and saturated zone were averages of 2
replications; other values represent single determinations.

b I I I I I I I I I
Negative Sign indicates decrease in percent, p081t1ve Sign
indicates increase in per cent.

CMedian ratio was selected from this location.

Nitrate-N/chloride ratios increased 49 and 124 per
cent as the experimental solution in the profiles moved from
the 0-10 cm increment to the 10-30 cm increment of the
South and North BLWRS respectively. This indicates that

nitrate had entered the experimental solution percolate.

49

Nitrification within these increments was occurring at a
rate greater than the rate of downward movement of the
experimental solution front and/or greater than rate of
denitrification. Aeration status may have been such that
nitrification was promoted while denitrification was
slowed. Data obtained by Erickson 35 El (1974), show that
oxidation-reduction potentials were higher in the South
BLWRS at the 30 cm depth compared to redox potentials at
the 15 cm depth (Table 14). Also, organic carbon content
of the soils below the surface would be lower; this would
help explain a decrease in denitrification. The occurrence
of nitrification is indicated by the observance that ratios
increased more in the North BLWRS than in the South BLWRS
which was well aerated before experimental solution was
applied.

Table 14. Oxidation-reduction potential measurements made in

the Swine South BLWRS during the Summer of 1972 in
millivolts (Erickson et 31, 1974).

 

 

Date 6/28 7/5 *7/28 8/1

Depth

15 cm 135 160 130 210
213 180 140 130

30 cm 180 190 240 250
260 215

120 cm 20 120 165 250
160 170 180 230

150 an -150 -140 - 20 -120
-170 -130 - 4o - 40

180 cm -160 -170 -105 - 80

-100 -l30 - 60 - 4O

 

50

After peaking near the 30 cm depth, the nitrate-N/
chloride ratios decreased 9 and 39 per cent as the solution
passed through the 30-152 cm increment of the South and
North BLWRS respectively. This indicates that denitrifi-
cation was occurring even at lower depths in the BLWRS. As
mentioned earlier, organic carbon seems to be accumulating
in the profile even at lower depths; therefore an organic
substrate is present to allow some denitrification to occur.

Ratios in the South BLWRS then decreased 19 per cent as
the solution passed from the 30-152 cm increment of the
profile into the saturated zone at the control site. This
indicates that the saturated zone by being anaerobic provided
an environment conducive to denitrification. Additional
sources of carbon were found to increase amount of
denitrification since the ratio decreased 47 per cent as the
solution moved through the molasses treatment compared to
19 per cent in the control treatment. Decreases in ratios
also occurred as the experimental solution passed through
the molasses and cracked corn treatment sites in the North
BLWRS.

The nitrate-N/chloride ratios of the tile effluents
also reveal the importance of additions of carbon in the
saturated zone. The ratio of tile effluent beyond the
control site was 1.51 as compared to 1.41, 0.49 and 1.21,
which were ratios of the experimental solution after the
solution had passed through molasses, molasses, and corn

treatment sites respectively.

51

Nitrate-N/chloride ratios in water samples taken from
tile effluent (RT) were used in computing percentage of
nitrate removed by the BLWRS. The nitrate-N/chloride ratio

of the experimental solution (RE) was assigned a value of

100 per cent and then multiplied by the fraction (B§§%_EZ).

For example, per cent nitrate removed by the South BLWRS

2.32 - 1.51
2.32

Other percentages of nitrate removed were 41 per cent,

= 35%.

 

at the control treatment end equalled 100% x

50 per cent, and 78 per cent by the North molasses, North
cracked corn and South molasses treatment ends respectively.
An explanation for the disparity between the two values

for molasses treatments may be that molasses was added to
the North BLWRS on the side farthest away from the tile
effluent whereas the molasses was added to the South BLWRS
on the side closest to the tile outlet. This means that in
the South BLWRS a higher concentration of organic carbon
most likely was present in the zone through which a major
part of the solution was passing through.

Data concerning the movement of the chloride peak in
the BLWRS are also shown in Tables 9-12. These data show
that chloride movement depended on the number of days waste
had been applied after application of the experimental solu-
tion.

The effect of various chloride concentrations on
nitrate analysis by electrode has been studied (Balasubraman-
ian and Kanehiro, 1974). In this study, errors less than or

equal to 14 per cent were reported when nitrate-N/chloride

52

ratios were between 0.10 and 1.00. Concentrations of
nitrate-N used in this study were 10 ppm or greater as was
true for most of the BLWRS samples. This error would
affect analysis of BLWRS samples whose ratios were in the
reported range. Analysis of samples, whose ratios were
greater than 1.00 would have been less affected because
Chloride interference is much less when its concentration
is less than nitrate-nitrogen.

Population counts of total anaerobes and denitrifiers
were conducted on the Swine BLWRS in November 1972
(Erickson 25.21, 1974). Data from this study (Table 15)
show that the largest populations of both anaerobes and
denitrifiers were in the surface soil. This is evidence
that considerable denitrification was occurring in the
microenvironments of the surface soil. Denitrifier popula-
tions were one to two orders lower in the anaerobic zone,
yet apparently this was sufficient biomass to allow nitrate
reduction to occur. Furthermore, the populations within the
anaerobic zone where carbon was added, were not assayed.
Populations in these environments could be expected to be
higher.

Some of the reduction of nitrate in the BLWRS can be
attributed to assimilatory nitrate reduction. In studies
conducted on the rich organic sediments in fresh water lakes
(Keeney 25.2lr 1971), it was determined that approximately
one-third of the nitrate-nitrogen passing through these

sediments was assimilated whereas two-thirds of the nitrate-

53

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54

nitrogen was denitrified. In light of this observation,
denitrification still assumes the major role in nitrate

removal in the BLWRS.

CONCLUSIONS

An experiment was conducted to determine amounts,
locations and environmental conditions of denitrification in
a Barriered Landscape Water Renovation System (BLWRS).
Solutions of nitrate and chloride were added to the BLWRS
surface and the ratio of concentration of nitrate-nitrogen
to concentration of chloride was monitored as the sclution
front moved through the BLWRS soil profile and out of the
BLWRS as tile effluent. Reduction in nitrate-N/chloride
ratio of the experimental solution as it passed through a
particular location was interpreted as evidence that
denitrification had occurred. Amounts of reduction in
nitrate-N/chloride ratio between various locations within
the North and South BLWRS are listed below.

1) Nitrate-N/chloride ratios decreased 44 and 47 per
cent as the solution passed through the upper 10 centimeters
of the soil profiles of the South and North BLWRS respective-
1y.

2) Nitrate-N/chloride ratios decreased 9 and 39 per
cent as the solution passed from the 10-30 cm increment to
the 30-152 cm increment of the profiles of the South and

North BLWRS respectively.

55

56

3) Nitrate-N/chloride ratios in the South BLWRS
decreased 19 per cent at the control site in the saturated
zone whereas the ratio decreased 47 per cent at the molasses
treatment site in the saturated zone. In the North BLWRS,
ratios of the experimental solution also decreased at the
molasses and corn treatment sites.

These data indicate that a significant amount of
denitrification occurred in the surface horizon of the
BLWRS. Denitrification also can be assumed to have
occurred in the lower portion of the aeration zone. A
significant amount of denitrification also was occurring
in the saturated zone. Increases in amount of denitrifica-
tion occurred in the saturated zone where organic carbon in

the form of either cracked corn or molasses had been added.

LITERATURE CITED

LITERATURE CITED

Alexander, Martin. 1965. Nitrification. p. 307-343. In
W. E. Bartholomew and F. E. Clark (ed.) Soil nitrogen.
Agron. No. 10. Amer. Soc. of Agron. Madison,
Wisconsin.

Balasubramanian, V. and Y. Kanehiro. 1974. Adaptability
of nitrate specific ion electrode for nitrate analysis
in tropical soils. Hawaii Agric. Exp. Sta. Bull.

Bolin, Bert. 1970. The carbon cycle. Scientific American
223 (3):124-135.

Bollag, J. M., M. L. Orcutt and B. Bollag. 1970.
Denitrification by isolated soil bacteria under
various environmental conditions. Soil Sci. Soc.
Amer. Proc. 34:875-879.

0! I!

Bonicke, R. and J. Kazda. 1970. Uber dos vorkommen von
Koh1enhydrat-nitritreduktasen in schnellnwachsenden
Mycobacteriumrarten und ihre bedeutung fur die
differenzierung dieser arten. Zentralbl. Bakteriol.
Parasitenk. Infiktions Kr. Hyg., Abt. I. 213:68-81.

 

Bonicke, R., S. E. Juhasc, and U. Diener. 1970. Studies
on the nitrate reductase activity of mycobacteria in
the presence of fatty acids and related compounds.
Amer. Rev. Resp. Dis. 102:507-515.

Bouwer, Herman. 1973. Renovating secondary effluent by
ground water recharge with infiltration basins.
p. 164-175. In W. E. Sopper and L. T. Kardos (ed.)
Recycling treated municipal wastewater and sludge
through forest and cropland. Pennsylvania State
University Press, University Park, Penn.

Bremner, J. M. 1965a. Total nitrogen. p. 1149-1178.
In C. A. Black, 35 21 (ed.) Methods of soil analysis.
Part II. Agron. No. 9. Amer. Soc. of Agron.
Madison, Wisconsin.

Bremner, J. M. 1965b. Inorganic forms of nitrogen.
p. 1179-1237. In C. A. Black et 31 (ed.) Methods of
soil analysis. Part II. Agron. No. 9. Amer. Soc.
of Agron. Madison, Wisconsin.

57

58

Broadbent, F. E. 1973. Factors affecting nitrification-
denitrification in soils. p. 232-244. In W. E.
Sopper and L. T. Kardos (ed.) Recycling treated
municipal wastewater and sludge through forest and
cropland. Pennsylvania State University Press,
University Park, Penn.

Campbell, N. E. R. and H. Lees. 1967. The nitrogen cycle.
p. 194-215. In A. D. McLaren and G. H. Peterson (ed.)
Soil Biochemistry Vol. I. Marcel Dekker, Inc., New
York.

Collins, F. M. 1955. Effect of aeration on the formation
of nitrate reducing enzymes by P. aeruginosa. Nature
175:173-174.

 

Cox, C. D. Jr., W. J. Payne and D. V. Dervartian. 1971.
Electron paramagnetic resonance studies on the nature
of hemeproteins in nitrate and nitric oxide reduction.
Biochim. Biophys. Acta 253:290-294.

DeGroot, G. N. and A. H. Stouthamer. 1970. Regulation of
reductase formation in Proteus mirabililis. Influence
of growth with azide and of haem deficiency on nitrate
reductase formation. Arch. Mikrobiol. 74:340-349.

 

Delwiche, C. C. 1967. Energy relationships in soil bio-
chemistry. In A. D. McLaren and G. E. Peterson (ed.)
Soil biochemistry. Marcel Dekker, Inc., New York.

Delwiche, C. C. 1970. The nitrogen cycle. Scientific
American 223 (3):136-147.

Dholakia, G. G., J. H. Stone and H. P. Burchfield. 1970.
Methanol requirement and temperature effects in waste-
water denitrification. Water Pollution Control Research
Series. U. S. E. P. A. water Quality Office, Washington,
D. C.

Downey, R. J. 1966. Nitrate reductase and respiratory
adaptation in Bacillus stearothermophilus. J.
Bacteriol. 91:634-641.

 

Downey, R. J. and D. F Kiszkiss. 1969. Oxygen and nitrate
induced modification of the electron transfer system of
Bacillus stearothermophilus. Microbios. 2:145-153.

 

Downey, R. J. and J. H. Nuner. 1967. Induction of nitrate
reductase under conditions of nitrogen depletion.
Life Sci. 6:855-861.

59

_DuToit, P. J., D. F. Toerien, and T. R. Davies. 1970.
Enzymatic patterns in two denitrifying microbial
populations. Water Res. 4:149-156.

Ellis, B. G. 1973. The soil as a chemical filter.
p. 46-70. In W. E. Sopper and L. T. Kardos (ed.)
Recycling treated wastewater and sludge through forest
and cropland. Pennsylvania State University Press,
University Park, Penn.

Erickson, A. E., B. G. Ellis, J. M. Tiedje, A. R. Wolcott,
C. M. Hansen, F. R. Peabody, E. C. Miller, and J.
W. Thomas. 1974. Soil modification for denitrifica-
tion and phosphate reduction of feedlot waste.
Environmental Protection Technology Series, EPA-660/2-
74-057, U. S. E. P. A.

Erickson, A. E., J. M. Tiedje, B. G. Ellis and C. M. Hansen.
1971. A Barriered Landscape Water Renovation System
for removing phdsphate and nitrogen from liquid feedr
lot waste. Proc., Int. symposium on Livestock Wastes,
Amer. Soc. of Agric. Eng.

Erickson, A. E., J. M. Tiedje, B. G. Ellis, and C. M.
Hansen. 1972. Initial observations of several medium
sized Barriered Landscape Water Renovation Systems for
animal wastes. Cornell Waste Management Conf. Proc.
(Ithaca, New York):405-410.

Focht, D. D. and H. Joseph. 1973. An improved method for
the enumeration of denitrifying bacteria. Soil Soc.
of Amer. Proc. 37:698-699.

Gruener, N. and H. J. Shuval. 1970. Health aspects of
nitrogen in drinking water. p. 89-105. In H. I.
Shuval (ed.) Developments in water quality research.
Ann Arbor - Humphrey Sci. Pub., Ann Arbor, Michigan.

Hart, S. A. and M. E. Turner. 1965. Lagoons for livestock
manure. Jour. Water Poll. Control Fed., 37:1578-1596.

International Rice Research Institute Annual Report. 1969.
p. 139-140.

Jansson, S. L. 1958. Tracer studies on nitrogen transfor-
mations in soil with special attention to mineraliza-
tion-immobilization relationships. Kungl. Lantbruk-
shogskolans Ann. 24:101—361.

Keeney, D. R., R. L. Chen and D. A Graetz. 1971. Importance
of denitrification and nitrate reduction in sediments
to the nitrogen budgets of lakes. Nature 233:66-67.

60

Klotter, H. E. 1970. Denitrification of ground water.
p. 93-140." In W. Husmann (ed.) Vom Wasser. Ein
Jabrbuch fur Wasserchemie und Wasserreinigungstechnik.
Verlag Cherrie GHBH, Weinbein-Berptr., West Germany.

Kolenbrander, G. J. 1972. Does leaking of fertilizers
affect the quality of ground water at the waterworks?
Stikstof 15:8-15.

Konishi, Y. 1969. Nitrite forming bacteria in Yamahaimato.
IV. Temperature sensitive nitrite reductase formation
in psychophilic nitrate respiring bacteria. J.
Ferment. Technol. 47:759-771.

Lance, J. C. 1972. Nitrogen removal by soil mechanisms.
J. Water Pollut. Cont. Fed. 44:1352-1361.

Lijinsky, W. and S. S. Epstein. 1970. Nitrosoamines as
environmental carcinogens. Nature 225:21-23.

McCarty, P. L. 1972. Energetics of organic matter
degradation. p. 91-118. In R. Mitchell (ed.) Water
pollution microbiology. Wiley-Interscience, New York.

McCarty, P. L., L. Beck and P. St. Amant. 1969. Biologi-
cal denitrification of agricultural wastewater by

addition of organic materials. Purdue Univ. Eng. Ext.
Ser. 135:1271—1285.

Meek, B. D., L. B. Grass and A. J. MacKensie. 1969.
Applied nitrogen losses in relation to oxygen status
of soils. Soil Sci. Soc. of Amer. Proc. 33:575-578.

Miller, R. H. 1973. The soil as a biological filter.
p. 71-94. In W. E. Sopper and L. T. Kardos (ed.)
Recycling treated municipal wastewater and sludge
through forest and cropland. Pennsylvania State
University Press, University Park, Penn.

Ngoddy, P. O. and J. P. Harper, R. K. Collins, G. D. Wells
and F. A. Heidar. 1971. Closed systemwwaste management
for livestock. Water Poll. Control Res. Series, U. S.
E. P. A.

O'Callaghan, J. R., V. A. Dodd and K. A. Pollock. 1973.
The long term management of animal manures. J. Agric.
Eng. Res. 18:1-12.

Payne, W. J. 1970. Energy yields and growth of heterotrophs.
Annu. Rev. Microbiol. 24:17-52.

. 1973. Reduction of nitrogenous oxides by
microorganisms. Bacteriological reviews 37:409-452.

 

61

Pichinoty, F. and L. D'Ornano. 1961. Inhibition by
oxygen of biosynthesis and activity of nitrate-
reductase in aerobacter aerogenes. Nature 191:
701-711.

 

Postgate, J. R. (ed.). 1971. The chemistry and biochemistry
of nitrogen fixation. Plenum Pub. Co., New York.

Provasoli, Luigi. 1970. Algal nutrition and eutrophication.
p. 574-593. In Eutrophication: causes, consequences,
correctives. Proc. of a Symposium, National Academy
of Sciences, Washington, D. C.

Schnitzer, M. and S. U. Khan. 1972. Humic substances in
the environment. Marcel Dekker, Inc., New York.

Schulp, J. A. and A. H. Stouthamer. 1970. The influence of
oxygen, glucose, and nitrate upon the formation of
nitrate reductase and the respiratory system in
Bacillus licheniformus. J. Gen. Microbiol. 73:95-112.

 

Showe, M. K. and J. A. DeMoss. 1968. Localization and
regulation of synthesis of nitrate reductase in
Escherichia coli. J. Bacteriol. 95:1305-1313.

 

Simoni, R. D. and M. K. Shullenberger. 1972. Coupling
of energy to active transport of amino acids in
Escherichia coli. Proc. Nat. Acad. Sci. U. S. A.
69:2663-2667.

 

Sinclair, P. R. and D. C. White. 1970. Effect of nitrate,
fumarate, and oxygen on the formation of the membrane
electron transport system of Haemophilus parainfluenza.
J. Bacteriol. 101:365-372.

 

Skoog, A. and D. M. West. 1969. Fundamentals of analytical
chemistry. 2nd edition. Holt, Rinehart and Winston,
Inc., New York.

Sopper, W. E. and L. T. Kardos. 1973. Vegetation responses
to irrigation with treated municipal wastewater. p. 271-
294. In W. E. Sopper and L. T. Kardos (ed.) Recycling
treated municipal wastewater and sludge through forest
and cropland. Pennsylvania State University Press,
University Park, Penn.

Sperl, G. T. and D. S. Hoare. 1971. Denitrification with
methanol. Selective enrichment for Hypomicrobium
species. J. Bacteriol. 108:733-736.

 

Stefanson, R. C. 1972. Soil denitrification in sealed
soil-plant systems. Plant Soil 37:113-149.

62

Stevenson, F. J. 1965. Origin and distribution of nitrogen
in soil. p. 1-42. In W. V. Bartholomew and F. E.
Clark (ed.) Soil nitrogen. Agron. No. 10. Amer. Soc.
of Agron. Madison, Wisconsin.

Taiganides, E. P., T. E. Hazen, E. R. Baumann, and D.
Johnson. 1964. Properties and pumping characteristics
of hog wastes. Transactions, Amer. Soc. Agric. Eng.
7:123.

Thomas, R. E. 1973. The soil as a physical filter. p. 38-
45. In W. E. Sopper and L. T. Kardos (ed.) Recycling
treated municipal wastewater and sludge through forest
and cropland. The Pennsylvania State University Press,
University Park, Penn.

U. S. Department of Health, Education and welfare. 1962.
Public Health Service drinking water standards. U. S.
Dept. Health, Educ. and Welfare Pub. 956.

Viets, F. G. Jr. and R. H. Hageman. 1971. Factors affect-
ing the accumulation of nitrate in soil, water and
plants. USDA Agriculture Handbook No. 413.

Von Mechsner, K. and K. Wahrmann. 1963. Beitrag zur
kenntris der mikrobiellen denitrifikation. Pathol.
Microbiol. 26:579-591.

WOlcott, A. R. 1972. Carbon and nitrogen reactions in the
soil. p. 44-63. In Impact of wastewater on soils.
Institute of Water Research, Michigan State University,
East Lansing, Michigan.

Wolcott, A. R. 1974. Personal communication.
Woldendorp, J. W., K. Dilz and G. J. Kolenbrander. 1966.

The fate of fertilizer nitrogen on permanent grassland
soils. Proc. Gen. Mtg. Europ. Grassland Fed. 1965:53-76.

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