PB-237 817
MICROBIOLOGY OF SEWAGE SLUDGE DISPOSAL IN SOIL
Robert H. Miller
Ohio Agricultural Research and Development Center
Prepared for:
National Environmental Research Center
November 1974
Rlgfon 18 Library
Environmental Protection ferny
DISTRIBUTED BY:
National Technical Information Service
U. S. DEPARTMENT OF COMMERCE
-------
TECHNICAL REPORT DATA
(Please read instructions on the reverse before comptetinrl
1. REPORT NO.
EPA-670/2-74-074
2.
PB 237 817
4. TITLE AND SUBTITLE
MICROBIOLOGY OF SEWAGE SLUDGE DISPOSAL IN SOIL
REPORT DATE ,
November 1974; Issuing Date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Robert H. Miller
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Ohio Agricultural Research and
Development Center
Wooster, Ohio 44691
10. PROGRAM ELEMENT NO.
1BB043; ROAP 21-ASE; Task 005
1. CONTRAC
14-12-824
12. SPONSORING AGENCY NAME AND ADDRESS
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPEOF REPORT AND PERIOD COVERED
Final .
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
PRICES SUBJECT TO CHANGE
16. ABSTRACT
Laboratory studies were conducted to evaluate some of the factors which
influence the microbial degradation of anaerobically digested sewage sludge in soils
and the population of microorganisms involved in these processes. Anaerobically
digested sewage sludge was rather resistant to decomposition with a maximum of about
20% of the sludge carbon evolved as COI in 6 months. The rate of decomposition at the
high loading rates of 90 and 224 metric tons/ha of dry solids was found to be independ-
ent of differences in soil chemical properties. Differences in soil texture influenced
sludge decomposition indirectly by influencing soil aeration under saturated moisture
conditions. A relationship was shown between the percent sludge carbon evolved as C0_
and Monthly Degree Days which will provide a method for predicting the amount of sludge
decomposition in a given climatic area based on available temperature data. Accumula-
tion of soluble soil nitrogen and soluble salts in sludge amended soils could limit the
rate of application sewage sludge to soils. Analyses of the soil solution coupled with
plant analyses of Kentucky 31 Fescue have shown that the solubility and plant uptake
of some metal ions is increased appreciably, primarily by the lowering of pH associated
with nitrification of sludge nitrogen. Both fungi and bacteria increased in numbers
in response to sludge amendments. Detailed characterization of bacterial isolates
showed definite changes in the morphology and physiology of bacteria from sludge
amended soils.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
*Sludge disposal, *Soils, Microbiology,
^Metabolism, Trace elements, *Sewage, Land
reclamation, Water quality, Indicator
species, Nitrogen, Phosphorus, Potassium,
Sodium, Calcium, Magnesium, Zinc, Copper,
Boron, Manganese, *Sludge drying
*Sewage sludge, "Sludge
decomposition, *CO_
evolution, *Sludge appli-
cation rate, Bacterial
pathogens, Enzymatic
activity, Soil humus,
Biochemical activity,
Phytotoxicity
13B
8. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
EPA Form 2220-1 (9-73)
20. SECURITY CLASS (Thispage)
Unclassified
1 ""NATIONAL TECHNICAL
'INFORMATION SERVICE
U S Department of Commerce
Springfield VA 22151
21. NO. OF PAGES
-------
REVIEW NOTICE
The National Environmental Research Center--
Cincinnati has reviewed this report and approved
its publication. Approval does not signify that
the contents necessarily reflect the views and
policies of the U. S. Environmental Protection
Agency, nor does mention of trade names or com-
mercial products constitute endorsement or recom-
mendation for use.
ii
-------
FOREWORD
Man and his environment must be protected from the adverse effects
of pesticides, radiation, noise and other forms of pollution, and the
Unwise management of solid waste. Efforts to protect the environment ,
require a focus that recognizes the interplay between the components
of our physical environment~-air, water, and land. The National
Environmental Research Centers provide this multidisciplinary focus
through programs engaged in:
studies on the effects of environmental contaminants on man and
the biosphere, and
a, search for ways to prevent contamination and to recycle valuable
resources.
The research reported here was conducted for the Ultimate Disposal
Section of the Advanced Waste Treatment Research Laboratory to determine
soil and climatic factors that affect biological decomposition of di-
gested sewage sludge in soils. Better selection of sludge spreading
sites and management of sludge amended soils should result from the
use of the information contained in the report.
A. W, Breidenbach, Ph.D.
Director
National Environmental
Research Center, Cincinnati
iii
-------
ABSTRACT
Laboratory studies were conducted to evaluate some of the factors which
influence the microbial degradation of anerobically digested sewage
sludge in soils and the population of microorganisms involved in these
processes.
Anaerobically digested sewage sludge was rather resistant to decompo-
sition with a maximum of about 20% of the sludge carbon evolved as C02
in 6 months. The rate of decomposition at the high loading rates of
90 and 224 metric tons/ha of dry solids was found to be independent bf
differences in soil chemical properties. Differences in soil texture
influenced sludge decomposition indirectly by influencing soil aeration
under saturated moisture conditions. A relationship was shown between
the percent sludge carbon evolved as C0ซ and Monthly Degree Days which
will provide a method for predicting the amount of sludge decomposition
in a given climatic area based on available temperature data.
Accumulation of soluble soil nitrogen and soluble salts in sludge amended
soils could limit the rate of application of sewage sludge to soils.
Analyses of the soil solution coupled with plant analyses of Kentucky
31 Fescue have shown that the solubility and plant uptake of some metal
ions is increased appreciably, primarily by the lowering of pH assoc-
iated with nitrification of sludge nitrogen.
Both fungi and bacteria increased in number in response to sludge
amendments. Detailed characterization of bacterial isolates showed
definite changes in the morphology and physiology of bacteria from
sludge amended soils.
This report was submitted in fulfillment of Contract No. 14-12-824,
under the sponsorship of the Office of Research and Development,
Environmental Protection Agency.
iv
-------
CONTENTS
Page
Abstract iv
List of Figures vl
List of Tables x
Acknowledgements xiii
Sections
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Materials and Methods 5
V Results and Discussion 17
Phase 1 Measurement of C$2 Evolution 17
Phase 2 Microbiological Study 32
Phase 3 Analyses of the Displaced Soil Solutions 63
Phase 4 Effects of Anaerobically Digested Sewage 98
Sludge on Kentucky 31 Fescue
VI References 115
VII Appendix A 118
-------
FIGURES
No. Page
1 Assembled C02 train with manifold, soil columns, 12
and bubble towers
2 Cumulative plot of C02~C evolution from sludge amended 18
soils during temperatures equivalent to autumn-winter
(Columbus, Ohio)
3 Cumulative plot of COo-C evolution from sludge amended
soils during temperatures equivalent to winter-spring
(Columbus, Ohio)
4 Cumulative plot of C02~C evolution from sludge amended 21
soils during temperatures equivalent to spring-summer
(Columbus, Ohio)
5 Cumulative plot of C02-C evolution from sludge amended 22
soils during temperatues equivalent to summer-autumn
(Columbus, Ohio)
6 Relationship between 7ป sludge carbon evolved as C02 and 23
degree days (90 metric ton/ha amendment)
7 Relationship between % sludge carbon evolved as C02 and 24
degree days (224 metric ton/ha amendment)
O ฃ
8 Cumulative plot of CO^C evolution from sludge amended
Ottokee sand (autumn-winter temperatures, Columbus, Ohio)
9 Cumulative plot of COj-C evolution from sludge amended 27
Celina silt loam (autumn-winter temperatures, Columbus,
Ohio)
10 Cumulative plot of C02~C evolution from sludge amended 28
Paulding clay (autumn-winter temperatures, Columbus, Ohio
11 Dilution plate counts of bacteria and actinomycetes in 33
sludge amended Ottokee sand as influenced by loading rate,
soil moisture content, time of incubation and temperature
of incubation ( Experiments I-IV)
12 Dilution plate counts of bacteria and actinomycetes in 34
sludge amended Celina silt loam as influenced by loading
rate, soil moisture content, time of incubation and tem-
perature of incubation (Experiment I-IV)
vi
-------
No. Page
13 Dilution plate counts of bacteria and actinomycetes in 35
sludge amended Paulding clay as influenced by loading
rate, soil moisture content, time of incubation and
temperature of incubation (Experiments I-IV) '
14 Dilution plate counts of anaerobic bacteria in sludge 37
amended Ottokee sand as influenced by loading fate, soil
moisture content, time of incubation and temperature
of incubation (Experiments I-IV)
15 Dilution plate counts of anaerobic bacteria in sludge 38
amended Celina silt loam as influenced by loading rate,
soil moisture content, time of incubation and temperature
of incubation (Experiments I-IV)
16 Dilution plate counts of anaerobic bacteria in sludge 39
amended Paulding clay as influenced by loading rate,
soil moisture content, time of incubation and temperature
of incubation (Experiments I-IV)
17 Dilution plate counts of soil fungi in sludge amended ^0
Ottokee sand as influenced by loading rate, soil moisture
content, time of incubation, and temperature of incubation
(Experiments I-IV)
18 Dilution plate counts of soil fungi in sludge amended ^
Celina silt loam as influenced by loading rate, soil
moisture content, time of incubation, and temperature
of incubation ( Experiments I-IV)
19 Dilution plate counts of soil fungi in sludge amended ^^
Paulding clay as influenced by loading rate* soil moisture
content, time of incubation, and temperature of incubation
(Experiments I-IV)
20 Soil pH in incubated columns of sludge amended and control "
Ottokee sand (Experiments I-IV)
21 Soil pH in incubated columns' of sludge amended and control -'o
Celina silt loam (Experiments I-IV)
22 Soil pH in incubated columns of sludge amended and control -*9
Paulding clay (Experiments I-IV)
23 Relationship of solution pH to soil pH (Ottokee sand) 60
24 Relationship of solution pH to soil pH (Celina silt loam) ^1
vil
-------
No. Page
25 Relationship of solution pH to soil pH (Paulding clay) 62
26 Soluble nitrogen in displaced soil solutions from sludge 71
amended and control Ottokee sand incubated at field
capacity (Experiment I, autumn-winter)
27 Soluble nitrogen in displaced soil solutions from sludge 72
amended and control Ottokee sand incubated under sat-
urated conditions (Experiment I, autumn-winter)
28 Soluble nitrogen in displaced soil solutions from sludge 73
amended and control Ottokee sand incubated at field ca-
pacity (Experiment II, winter-spring)
29 Soluble nitrogen in displaced soil solutions from sludge 74
amended and control Ottokee sand incubated under saturated
conditions (Experiment II, winter-spring)
30 Soluble nitrogen in displaced soil solutions from sludge 75
amended and control Ottokee sand incubated at field capacity
(Experiment III, spring-summer)
31 Soluble nitrogen in displaced soil solutions from sludge 76
amended and control Ottokee sand incubated under saturated
conditions (Experiment III, spring-summer)
32 Soluble nitrogen in displaced soil solutions from sludge 77
amended and control Ottokee sand incubated at field ca-
pacity (Experiment IV, summer-autumn)
33 Soluble nitrogen in displaced soil solutions from sludge 78
amended and control Ottokee sand incubated under saturated
conditions (Experiment IV, summer-autumn)
34 Soluble nitrogen in displaced soil solutions from sludge 79
amended and control Celina silt loam incubated at field
capacity (Experiment I, autumn-winter)
35 Soluble nitrogen in displaced soil solutions from sludge 80
amended and control Celina silt loam incubated under
saturated conditions (Experiment I. autumn-winter)
36 Soluble nitrogen in displaced soil solutions from sludge 81
amended and control Celina silt loam incubated at field
capacity (Experiment II, winter-spring)
37 Soluble nitrogen in displaced soil solutions from sludge 82
amended and control Celina silt loam incubated under
saturated conditions (Experiment II, winter-spring)
38 Soluble nitrogen in displaced soil solutions from sludge 83
amended and control Celina silt loam incubated at field
capacity (Experiment III, spring-summer)
viii
-------
No. Page
39 Soluble nitrogen in displaced soil solutions from sludge 84
amended and control Celina silt loam incubated under
saturated conditions (Experiment III, spring-summer)
40 Soluble nitrogen in displaced soil solutions from sludge 85
amended and control Celina silt loam incubated at field
capacity (Experiment IV, summer-autumn)
41 Soluble nitrogen in displaced soil solutions from sludge 86
amended and control Celina silt loam incubated under
saturated conditions (Experiment IV, summer-autumn)
42 Soluble ammonium and nitrate nitrogen in displaced soil 87
solutions from sludge amended and control Paulding clay
(Experiment I, autumn-winter)
43 Soluble ammonium and nitrate nitrogen in displaced soil 88
solutions from sludge amended and control Paulding clay
(Experiment II, winter-spring)
44 Phosphorus content of Kentucky 31 Fescue grown in sludge 104
amended soils
45 Potassium content of Kentucky 31 Fescue grown in sludge 106
amended soils
46 Calcium in content of Kentucky 31 Fescue grown in sludge 107
amended soils
47 Magnesium content of Kentucky 31 Fescue grown in sludge 108
amended soils
48 Sodium content of Kentucky Fescue grown in sludge amended 109
soils
49 Zinc content of Kentucky 31 Fescue grown in sludge amended 110
soils
50 Copper content of Kentucky 31 Fescue grown in sludge 11-1
amended soils
51 Boron content of Kentucky 31 Fescue grown in sludge amended 112
soils
52 Manganese content of Kentucky 31 Fescue grown in sludge 114
amended soils
ix
-------
TABLES
No. . Page
1 Research plan: Microbiology of sewage sludge 6
decomposition in soils
2 Temperature program for sludge disposal study 7
3 Physical and chemical properties of experimental 8
soils
4 Analyses of digested sewage sludge from Columbus 9
Jackson Pike Plant (three sampling times)
5 Analyses of freeze dried anaerobically digested 10
sewage sludge
6 Characters for which all bacteria were examined 14
7 Sludge decomposition in Ottokee sand as affected 29
by seasonal temperatures
8 Sludge decomposition in Celina silt loam as affected 30
by seasonal temperatures
9 Sludge decomposition in Paulding clay as affected 31
by seasonal temperatures
10 Relative survival of indicator bacteria in soils 44
amended with anaerobically digested sewage sludge
11 Distribution of bacterial isolates obtained from 46
dilution plating
12 Morphological and cultural characteristics'of 48
bacterial isolates from the Ottokee sand and
Celina silt loam soils ( Experiments I & II)
13 Morphological characteristics of bacterial isolates 49
from the Ottokee sand and Celina silt loam soils
(Experiments III & IV)
14 Morphological characteristics of bacterial isolates 50
from Paulding clay soils (Experiments I-IV)
15 Growth characteristics of bacterial isolates 51
16 Biochemical and enzymatic activity of bacterial 52
isolates
17 Acid production from selected carbohydrates (aerobic) 53
-------
No. Page
18 Acid production from selected carbohydrates 54
(anaerobic)
19 Sensitivity of bacterial isolates to seven 55
selected antibiotics
20 Specific conductance of soil solutions displaced 65
from sludge amended Ottokee sand
21 Specific conductance of soil solutions displaced 66
from sludge amended Celina silt loam
22 Specific conductance of soil solutions displaced 67
from sludge amended Paulding clay
23 Relationship of crop response to soil salinity 64
expressed in terms of the conductivity of the
saturation extract (Richards, 1954)
24 Organic matter contents of soil solutions displaced 69
from sludge amended soils
25 Percentage of the organic nitrogen of anaerobically 90
digested sewage sludge appearing in the soil solution
after 6 months incubation
26 Concentration of Ca in displaced soils solutions from 92
sludge amended soils
27 Concentration of Mg in displaced soil solutions from 93
sludge amended soils
28 Concentration of Na in displaced soil solutions from 94
sludge amended soils
29 Concentration of Zn in displaced soil solutions from 95
sludge amended soils
30 Concentration of Cu in displaced soil solutions from 96
sludge amended soils
31 Concentration of Mh in displaced soil solutions from 97
sludge amended soils
32 Effect of anaerobically digested sewage sludge on 99
germination of Kentucky 31 Fescue in sludge amended
soils
33 Effect of anaerobically digested sewage sludge on dry 101
matter yield of Kentucky 31 Fescue. Data expressed as
ratio, Sludge treatment/Control
xi
-------
No. Page
34 Effect of anaerobically digested sewage sludge on dry 102
matter yield of Kentucky 31 Fescue. Data expressed as
the mean of dry weight (g) for the 1, 3 and 6 months
incubation
35 Nitrogen and sulfur content of Kentucky 31 Fescue 103
grown in sludge amended soils
xii
-------
ACKNOWLEDGEMENTS
The author would like to acknowledge the technical assistance
of Mr. Dennis Zaebst, Mrs. Phyllis Socha, and Mrs, Hilde Burab
during all or part of this study.
Appreciation is also expressed for the administrative assistance
provided by the Ohio Agricultural Research and Development Center
during the.duration of the contract.
Finally, the author recognizes the significant contributions of
our departmental secretaries, Mrs. Gloria North and Mrs. Neima
Weate in typing the draft and final copy of this contract report
and to Mrs Martha Fockler for her drafting of many of the Figures
contained in this report.
xiii
-------
CONCLUSIONS
1. The organic compounds in anaerobically digested sewage sludge are
rather resistant to further microbial degradation in soil. Thus
land disposal of sewage sludge would be expected to increase the
organic matter content of the soil and markedly change its physical
and chemical properties.
2. The rate of sewage sludge decomposition at the high loading rates
used in this study (90 & 224 metric tons/ha) is largely independent
of soil chemical properties. Differences in soil pH, initial fer-
tility, organic matter content, nitrogen content, etc. would hot
influence the degradative activity of the microbial population.
3, Soil texture influences sludge decomposition indirectly by in-
fluencing soil aeration under saturated moisture conditions.
Anaerobiosis with an accompanying large decrease in the rate of
sludge decomposition, an increase in odor, and reducing conditions
were readily apparent in water saturated fine textured soils.
4. Soil temperature markedly affects the rate of sewage sludge de-
composition in soil. A relationship between per cent sludge carbon
evolved as CO and temperature, expressed as Monthly Degree Days
should be useful in predicting the magnitude of sludge decomposition
in a given season or climatic area.
5. A comparison of population changes for fungi and bacteria in re-
sponse to sludge amendments would indicate that both groups of
microorganisms can participate in sludge decomposition. The dom-
inant group under a particular set of environmental conditions
will depend on the soil moisture content and a degree of soil
aeration.
6. The population of total coliforms, fecal coliforms and fecal
streptococci added with the sludge decreased rapidly with time.
However, low numbers of total coliforms and fecal streptococci
were still detected in sludge amended soils after 6 months in-
cubation.
7. A total of 354 bacterial isolates from the Ottokee sand and
Celina silt loam soils were extensively characterized using
morphological and biochemical characters. The bacteria from
sludge amended soils had an increased tendency to be gram
negative, smaller, produce pigments, have a faster growth rate,
grow at higher salt concentrations, to be more resistant to
antibiotics, and to be less active in biochemical transformations.
8. Accumulation of soluble salts and soluble nitrogen in sludge
amended soils are the two factors most likely to limit the rate
at which sewage sludge may be added to soils.
-------
RECOMMENDATIONS
Futher studies should be carried out on the rate of sludge decomposition
with repeated small loadings, rather than with one massive loading such
as was employed in this study. Rates of decomposition should also be
determined for surface applied sludge versus that incorporated with the
soil and filter cake sludge vs liquid sludge vs freeze dried sludge.
Continued survival of low populations of indicator bacteria for periods
up to 6 months in sludge amended soils suggests that further studies
are needed on the survival of bacterial pathogens in soil. Even more
of an unknown is the fate of enteric viruses associated with digested
sewage sludge. Further studies should delineate the methodology for
investigating the survival of enteric viruses in soils.
The large accumulation of soluble nitrogen in sludge amended soils
points out the need for further studies on ways to accelerate losses
of nitrogen during handling, storing and applying sludge to land.
Such losses of nitrogen would greatly improve the suitability of
sludge for disposal on land.
-------
INTRODUCTION
Interest in land disposal of sewage sludges and primary and secondary
effluents from treatment of municipal and industrial wastes is growing
rapidly. The extent of this interest might be surmised by the increased
generation of literature and reports on this subject (Hinesly and
Sosewitz, 1969; Ewing and Dick, 1970; Hinesly jet al, 1971; CRREL Special
Report 171, 1972). To some, this approach to waste treatment or ultimate
disposal may seem like a new concept, but in actuality the disposal of
human wastes on land is as antiquated as man himself. Land disposal of
municipal wastes af.ter some degree of sanitary treatment has been em-
ployed by many European cites on a continuing basis for 50-100 years.
In this country a surprising number of smaller communities have used
land for disposal of sewage sludges and effluent for many years with
varying degrees of success (anonomyous, 1962).
The primary reason for the attractiveness of land disposal of liquid
sludge can probably be summed up in one wordeconomics. Recent cost
evaluations have shown the cost for disposal of liquid digested sewage
sludge on land within reasonable proximity to the treatment plant is
usually far less than any other type of disposal method e.g. drying
beds, lagoons, or incineration (Ewing and Dick, 1970; Burd, 1968). A
secondary reason for increased use of land disposal for liquid sludge
is that soil provides an alternate approach to the use of our already
stressed air and water resources for sludge disposal.
The studies funded by this contract have considered in some detail the
microbiological aspects of sewage sludge disposal in soil. The sig-
nificance of studies of this type can be shown by a brief evaluation
of the number of potential problems in land disposal of sewage sludge
which are related to microbial activity. One problem associated with
management qf soils for sludge disposal is the rate at which sludge
organic carbon compounds are decomposed in soil. Accumulation of organic
matter in soil can have both beneficial and detrimental effects on the
physical and chemical properties of soils. Information on rates of
sludge degradation can therefore be useful in modifying loading rates
and management to achieve the desired accumulation of organic matter.
Microbial activity during sludge decomposition also modifies the mo-
bility and solubility of inorganic compound and ions in soil by pro-
cesses of mineralization, immobilization, oxidation, reduction, and
chelation reactions. The impact of microbial reactions are particularly
significant in modifying the rate of sewage nitrogen by the processes
of ammonification, nitrification, and biological denitrification. One
of the long term problems with sludge disposal on land is the accumulation
of phytotoxic levels of heavy metal ions. Again, both the mobility and
plant uptake of these ions are influenced greatly by microbial activity
and the processes listed above. Unless treated, sewage sludge may
contain relatively large numbers of pathogenic bacteria and viruses.
Application of sewage sludge to agricultural lands or to lands being
reclaimed must not pose a health hazard to the surrounding community.
Again the normal microbial population of soil is involved in the rapid
die back of these pathogens and information on these reactions is of
considerable significance. Finally, microorganisms have been known to
produce numerous phytotoxins, most often when supplied with high levels
-------
of organic substrate. Because the additions of sewage sludge to soil
will increase the substrate level in soils, information must be obtained
on this potential problem.
This contract has addressed itself in varying detail to the problems
listed above. It is hoped that the information provided will increase
the understanding of all the myriad microbial reactions which influence
the success of a system for land disposal of sewage sludge.
-------
MATERIALS AND METHODS
An overview of the research plan for this study is shown in Table 1.
The experimental variables included three soils, Ottokee sand, Celina
silt loam, and Paulding clay; two sludge amendments, 90 tons (metric)/
ha (40 tons/acre) and 224 tons (metric)/ha (100 tons/acre) on a dry
weight basis; two soil moisture contents, field capacity ( 1/3 bar
moisture 7ป) and saturation; and temperature. Temperatures were programmed
within ah environmental control room to provide both diurnal and seasonal
temperature variation as shown in Table 2. Temperatures were maintained
at the minimum and maximum temperatures for 12 hours each day for one
month before changing to the next temperatures. The duration of each
experiment is shown by the horizontal lines beneath the temperature data
of Table 2 and corresponded to autumn-winter (Expo I), winter-spring
(Exp.II), spring-summer (Exp.III) and summer-autumn (Exp. IV) temperatures.
Detailed descriptions of the materials and methods employed will be provided
in the following paragraphs.
SOILS '
The three soils used in this study were chosen because they represented
the extremes in soil texture i.e. sand, silt and clay. Selected physical
and chemical properties are shown in Table 3. Bulk samples of each soil
were obtained from the 2-15 cm depth of each soil profile, sieved through
a 5 mm screeen to remove stones and plant debris, mixed and stored at
4ฐC until needed.
The Paulding clay and Celina silt loam soils were obtained from agri-
cultural land while the Ottokee sand was from a coniferous-hardwood
forest site. i
SEWAGE SLUDGE
Anaerobically digested sewage sludge was obtained from the Jackson Pike
Treatment Plant, Columbus, Ohio. A partial analysis of the liquid sludge
at three selected time periods are shown in Table 4.
Incorporation of liquid digested sewage sludge with soil in a single
application at the rates used in this study (90 and 224 ton (metric)/ha)
proved a difficult task. For this reason all sludge was freeze dried
prior to incorporation with the soil. The assumptions made when using
this approach was that freeze drying would not chemically alter the
sludge organic compounds nor influence the rate at which the microbial
population would decompose them once the sludge was incorporated with
the soil. Freeze drying may have altered the physical properties of the
sludge organic colloids, but the changes were considered similar to those
which would occur when sludge is dried in drying beds or after field
spreading.
Prior to freeze drying, fresh anaerobically digested sewage sludge was
concentrated to a slurry j.n vacuo at 40ฐC with a flash evaporator. The
slurry was frozen immediately in 30 x 43 mm stainless steel trays.
Trays containing the frozen sludge were transferred to the tray drying
-------
Table 1. RESEARCH PLAN; MICROBIOLOGY OF A SEWAGE SLUDGE DECOMPOSITION IN SOILS.
Variables: 1) Soil properties!) Rate of sludge amendment 3) Soil moisture content.
4) Temperature cycle
Plant Bioassay
(Kentucky 31
Fescue)
/ Phase 4
^Evaluate at
1,3,6 months
l)Germination
2)Yield
3) Plant analysis
Phase 3
Analysis of
Displaced
Soil Solutions
DpH
2)Conductivity
3)0rganic matter
4)0rganic N,.NH4"N, NO^-Nj
NO"-N
3
5)Nutrient analysis
(Emission Spectrograph)
12-Control
6-Field Capacity
6-Saturated
12-90 ton (metric)/ha
of sludge
6-Field Capacity
6-Saturated
12-224 ton (metric)/ha
6-Field Capacity
6-Saturated
36 columns/soil for
each experiment
Phase 1
6 Months
continuous
monitoring
^ C02 Evolution
Phase 2
Evaluate at
1,3, 6 months
Microbiological
Study
1) Total and fecal
coliforms and
fecal streptococci
2) Soil fungi
3) Anaerobic bacteria
4) Aerobic, heterotrophic
bacteria
a)Isolate and
characterize
b)Computer analysis
-------
Table 2. TEMPERATURE PROGRAM FOR SLUDGE DISPOSAL STUDY (AVERAGE MAXIMUM AND MINIMUM TEMPERATURES (ฐF) ARE
THOSE AT THE UNIVERSITY FARM, O.S.U., COLUMBUS, OHIO DATA 1894-1965).
Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May. Jun. Jul. Aug.
Avg. Max.
78.2 66.3 51.3 39.4 37.8 39.0 49.9 61.7 72.8 81.6 85.5 83.8
Avg. Min.
54.6 43.2 33.6 24.8 22.2 2207 31.2 40.5 50.3 59.3 63.0 61.3
EXP. I AUTUMN- WINTER
EXP.II WINTER-SPRING
EXP.Ill SPRING-SUMMER
EXP. IV AUTUMN EXP. iv SUMMER -
-------
Table 3. PHYSICAL AND CHEMICAL PROPERTIES OF EXPERIMENTAL SOILS
Organic Organic
Texture % C N Moisture %
Soil I Sand Silt Clay | 70 % pH | 0 Bar 1/3 Bar 15 Bar
Ottokee
Fine 95.5 1.3 3.2 0.5 0.025 7.1 20.0 3.1 2.6
Sand
00 Celina
Silt 17.9 63.9 18.2 1.6 0.080 7.0 35.0 21.7 9.6
Loam
Paulding
Clay 14.7 39.7 45.6 2.0 0.158 5.7 62.0 32.9 18.3
-------
Table 4. ANALYSES OF DIGESTED SEWAGE SLUDGE FROM COLUMBUS, JACKSON
PIKE PLANT (THREE SAMPLING TIMES) ALL VALUES ARE GIVEN AS mg/1.
Total N
Ammonia N
Total Solids
Ash
PH
Grease
P
K
Ca
Mg
Na
Mn
Fe
B
Cu
Zn
Al
Si
Ni
Cd
Pb
April 5,
1970
1316
357
47400
21100
7.3
4790
1670
120
.3340
1020
280
10
52
6
33
84
509
1280
1.1
0.5
4.2
April 20,
1970
1395
464
49100
20400
7.0
4820
1460
120
2630
870
290
11
49
4.8
34
75
453
1070
0.9
0.3
3.4
April 27,
1970
1234
536
35500
17130
7.1
...
1180
120
2240
240
150
7
67
3.5
10
58
215
2035
0.6
0.2
2.8
-------
Table 5. ANALYSIS OF FREEZE DRIED ANAEROBICALLY DIGESTED SEWAGE SLUDGE.
Exp.
#
I
II
III
IV
Organic
C
7o
25ป7
26.5
26.3
25.1
Organic
N
%
3.2
2.9
2.9
3.1
Free
NH~
%
0.17
0.21
0.32
0.23
C:N
%
7.6
8.5
8.2
7.5
Hexane
Soluble
%
8.9
8.3
8.6
7.8
Methanol
Soluble
%
4.5
4.7
4.5
4.5
Vola-
tiles
%
52.9
52.9
51.3
50.1
Ash
%
47.1
47.1
48.7
49.9
H20
%
4.3
5.5
4.0
3.9
25.9 3.0 0.23 8.0 8.4 4.5 51.8 48.2 4.4
-------
chamber of a Virtis Freeze Mobile for freeze drying,, Analyses of the
freeze dried sludge as used in the four basic experiments of this study
are shown in Table 5. It can be seen from these data that the carbon
and nitrogen components remained remarkably constant, even though these
analyses were of sewage sludge obtained periodically over a two year
period.
PHASE 1: MEASUREMENT OF C02 EVOLUTION
Carbon dioxide evolution from sludge amended soils was measured for
periods up to six months and used to determine the rate of decomposition
of sludge organic matter and as an indirect measure of microbial activity.
The basic apparatus for measuring CO- evolution was patterned after that
previously described by Stotzky (1965). Compressed air was delivered
from a pressure regulator to a scrubber system consisting of concentrated
sulfuric acid, AN NaOH, and distilled water in series; passed through a
glass gassing manifold with capillary tubing attached to equalized air
flow and over /the soil contained within plastic columns; then to a glass
bead bubble tower COo collectors containing IN NaOH.
The plastic columns which contained the soil were constructed from 20
mm lengths of 8 mm (I.D.) high impact styrene pipe. The bottom of each
column was sealed with a sheet of 6 mil polyvinylchloride (PVC) held in
place with Scotch Brand Super strength Adhesive and a few wraps of vinyl
electric tape. A No.14 rubber stopper containing glass air inlet and
outlet tubes 7 mm (I.D.) and 10 mm (I.D.) glass center tube was used to
close the top of each column. The center tube, used periodically to add
Hซ0 during the incubation, was closed with a rubber stopper.
All connections of the components of the COo apparatus were made with
tygon tubing. Each scrubber system serviced two manifolds with 14 outlets
in each. Two of the 14 outlets were used for control columns which did
not contain soil. The assembled apparatus including manifold, columns
and bubble towers are shown in Figure 1.
At the beginning of each experiment sufficient soil and freeze dried
sludge for all replications of a given treatment were mixed within a
twin shell blender for 30 minutes. Soil or soil-sludge mixtures equiva-
lent to 600 g of oven dry soil were added to each column and packed
gently by tapping. Distilled water was then added to the soil columns to
bring them to the desired moisture content and the columns closed with
the rubber stopper assembly described previously. All columns were then
transferred to the environmental control room and connected to the C02
apparatus.
Tumblers of the C02 collectors were placed regularly as needed with fresh
IN NaOH. Since microbial activity varied considerably during the different
incubation periods, the length of time between changes varied between one
day and two weeks. Evolved C02 absorbed within the C02 collectors was
determined by titration using a Beckman Model K Automatic Titrator after
precipitation of the carbonate with Ba Cl-.
11
-------
RffRODi:D315
Figure 1. Assembled CO,, train with manifold, soil columns, and bubble
tower C02 collectors.
12
-------
After 1, 3 and 6 months incubation duplicate columns of each soil were
removed from the CC>2 apparatus and used for the analyses listed under
Phases 2, 3 and 4 (Table 1) The soil samples removed from the columns
were thoroughly mixed, transferred to plastic lined cardboard cartons
(86 x 165 mm), and stored at 4ฐ C until analyzed. In most cases the
platings for the microbiological study, the displacement of the soil
solution, and platings for the plant bioassay were all completed within
one week. Storage at 4ฐC was utilized to minimize microbial changes be-
fore analysis.
PHASE 2: MICROBIOLOGICAL STUDY
Population estimates for a number of significant groups of microorganisms
in the sludge amended soils were made routinely after 1, 3 and 6 months
incubation. An attempt was made to perform all determinations within a
few days after the soil was removed from the columns to minimize changes
in the microbial population because of storage.
Population estimates of coliforms and fecal streptococci in sludge amended
soils were made from appropriate soil dilutions using membrane filter
techniques as described in Standard Methods (American Public Health
Association, 1965). Fecal coliforms were estimated by the high temperature
membrane filter technique developed by Geldreich _et al (1965) Fungi were
estimated by dilution plating on rose bengal-streptomycin agar (RBS)
(Martin, 1950). All plates were incubated for 7 days at 26ฐC before
counting.
Aerobic heterotrophic bacteria including actinomycetes were determined
by spreading 0.1 ml of the desired dilutions on pre-poured plates of
sludge-soil extract agar (SS)(See Appendix A.). All plates were incubated
for 7 days at 26ฐ C before counting. Anaerobic bacteria were also enumerated
on sludge-soil extract agar plates incubated in Gas-Pak Anaerobic jars
for two weeks at 26ฐC. Individual colonies of aerobic heterotrophic
bacteria for subsequent detailed characterization were isolated from
sludge-soil extract agar plates prepared from soils after 1 months in-
cubation. The isolated colonies were transferred into screw capped
nutrient agar slants and stored at 4ฐcฐ When possible the individual
colonies selected were taken from dilution plates containing 10 colonies
or less. Where this approach was not applicable, 10 colonies were selected
at random from the highest dilution series employed. All cultures were
transferred bi-monthly during the period of characterization.
Each of the cultures was examined for the morphological, colonial, phy-
siological and biochemical characters listed in Table 6. In preparation
for the various tests, stock cultures were transferrred to flasks of
nutrient broth. The inoculated flasks were shaken gently on a gyrotory
shaker until visible turbidity developed. These actively growing cultures
were then used for microscopic examination as well as for the source of
inoculum for the various test media. Standard techniques and media were
employed for the various biochemical tests (Collins & Lyne, 1970). Phy-
siological evolutions such as growth rate, temperature range, and growth
in NaCl were made on nutrient broth or nutrient agar. Acid production
from various carbohydrates was determined using the miniaturized Micro-
titer technique of Fung and Miller (1970). Acid production under
13
-------
Table 6. CHARACTERS FOR WHICH ALL BACTERIA WERE EXAMINED
Character
Cell shape
Cell length
Cell width
Presence of:
-spores
-plemorphism
-filaments
Cell arrangement
Gram Stain
Colony-size
" -shape
" -pigmentation
Type of growth
in broth
Relative growth
Mobility
Thermotolerance
Salt tolerance
Resistance to:
Viomycin
Streptomycin
Chloromycetin
Novobiocin
Bacitracin
Tetrocycline
Penicillin
Hydrolysis of:
-cellulose
-starch
-pectin
-gelatin
- tributyrin
-triolein
Production of:
-catalase
-cytochrome oxidase
-urease
-indole
-acetylmethylcarbinol
-acid (Methyl-red)
-hydrogen sulfide
Utilization of citrate
Litmus milk
Reduction of nitrate
No. of
States
5
3
3
2
2
2
4
3
3
8
9
4
3
2
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
7
4
Character
Production of acid from:
-glucose *
-fructose*
-mannose .*
-galactose *
-arabinose *
-ribose
-lactose *
-sucrose *
-maltose *
-cellobiose *
-raffinose
-dextrin
-inulin *
-mannitol
-sorbitol*
-dulcitol *
No. of
States
4
4
4
4
4
2
4
4
4
4
2
2
4
2
4
4
* Evaluated both aerobically and anaerobically.
L4
-------
anaerobic conditions also utilized the microtiter plates maintained in
Gas-Pak jars for a two week period. Incubation temperatures were 26ฐ C
except for the temperature studies and gelatin hydrolysis. Time of in-
cubation was variable for the various tests and took into account the
differing growth rates of the isolates.
Upon completion of the analyses, data were recorded on IBM punched cards
as a 1 for the presence of a character or a positive test, or as a 0
for the absence of a character or a negative test. Inapplicable characters
or tests not done were scored as a 3. All evaluations for each character
were expressed on a percentage basis by the use of a computer program.
PHASE 3: ANALYSIS OF DISPLACED SOIL SOLUTIONS
Soil solutions were displaced from the sludge amended and control soils
after 1, 3 and 6 months incubation. Solutions were obtained from the
Ottokee sand and Celina silt loam soils by a liquid displacement method
using 507o glycerol similar to that used by Larson and Widdowson (1968).
Moist soil, equivalent to 325 g of oven-dry soil, was mixed with 100 g
of acid washed quartz sand, and added stepwise with tamping to glass
columns. The columns were constructed of 100 x 2.5 cm lengths of pyrex
tubing closed at one end with a rubber stopper and 6 cm length of 7 mm
(I.D.) glass tubing. Glass wool was placed in the bottom of each column
to retain soil particles. Initial studies indicated that approximately
50% of the soil water could be collected without contamination with
glycerol. The time necessary to displace the desired quantity of soil
solution varied from about 1 hour in the Ottokee sand to as long as 12
hours with some samples of the Celina silt loam soil. Positive pressure
was applied in some instances to increase the flow rate through the
column. The volume of all solutions was measured and the solutions trans-
ferred to polyethylene bottles for storage at -20ฐ C. All solutions were
thawed rapidly in hot water before analysis. The liquid displacement
method was not effective in obtaining soil solutions from Paulding clay
because of very slow flow rates through the column. Soil solutions from
this soil were obtained by use of a pressure membrane at 15 atms. pres-
sure (Reitemeir and Richards, 1944). The pH of the soil solutions was
measured with a glass electrode using a Corning Model 10 pH Meter.
Specific conductance (mmhos/cm) was measured using an Industrial in-
struments, Model RC 16B2 Conductivity Bridge. The cell constant for the
conductance cell was 1.03.
Organic matter was determined colorimetrically after oxidation of the
organic matter with a 0.15 N potassium dichromate-sulfuric acid solution.
The resultant green color was read in a Coleman Universal Spectrophoto-
meter at 645 nm (Carolan 1948).
Organic nitrogen was measured by a micro technique described by Umbreit
_et al (1964). The digestion was carried out in 18 x 15 mm test tubes
placed within a heating block using a digestion mixture of 1 ml of 2N
sulfuric acid containing copper sulfate and sodium selenite. Ammonium
nitrogen in the digests was determined by Nesslerization.
Nitrate, nitrite and ammonium nitrogen were determined by the steam dis-
tillation methods of Bremmer (1965).
15
-------
Direct analysis of P, Ca, K, Mg, Na, Mn, Fe, Al, Zn, Cu, Mo, and B were
carried out on 5 ml aliquots of the soil solutions by use of a Jarrell-
Ash Model 66-000 direct reading Emission Spectrograph. *
PHASE 4: PLANT BIQASSAY (KENTUCKY 31 FESCUE)
Control and sludge amended soils were removed from the soil columns after
1, 3 and 6 months and sub-samples used to evaluate the effect of sewage
sludge on the germination, and dry matter yield of Kentucky 31 Fescue
(Festuca arundinacea) Fescue was chosen as the bioassay plant because
it is hardy and will grow reasonably well under saturated moisture con-
ditions.
Soil from each column equivalent to 225 g of oven dry soil was weighed
into 12 oz. waxed cardboard containers and compacted gently. Seventy
five seeds of Kentucky 31 Fescue were spread uniformly over the surface
of the soil in each container, and covered with 10 g of exfoliated ver-
miculite. Immediately after planting all of the containers were trans-
ferred to a growth chamber programmed for a 16 hour light period, a day
temperature of 30ฐC and a night temperature of 23ฐ C. The plants were
watered daily with distilled H 0 to maintain them at field capacity or
soil saturation.
Germination was evaluated 3 weeks after planting by counting the number
of developing seedlings. Dry matter yield of the top growth was deter-
mined 6 weeks after planting on plant material dried at 70ฐ C for 24
hours.
Plant samples were ground in a stainless steel Wiley Mill and subsamples
used for plant analysis. Potassium, phosphorus, calcium, magnesium, iron,
sodium, silicon, manganese, strontium, barium, boron, copper, zinc, moly-
bdenum and aluminum were determined by a direct reading Jarrell-Ash Model
66-000 Emission Spectrograph using the standard using the standard tech-
niques employed by the Ohio Plant Analysis Laboratory. Total nitrogen was
determined using the Technicon Kjeldahl Nitrogen Apparatus and modification
of the procedure of Ferrari. Sulfur was determined on perchloric-nitric
acid digests using a Ba SO^ turbidimetric technique.
" Ohio Plant Analysis Laboratory, Wooster, Ohio
16
-------
RESULTS AND DISCUSSION
PHASE 1: MEASUREMENT OF C02 EVOLUTION
Anaerobically digested sewage sludges contain about 25% organic carbon on
a dry weight basis (Burd, 1968), See also Table 5. During the process of
anaerobic digestion the waste organic solids are stabilized by the almost
complete microbial fermentation of carbohydrates (the exception is cell-
ulose) resulting in a 60-75% reduction in volatile solids. Although data
on the organic analysis of anaerobically digested sludge is difficult to
obtain the residual organic material consists of a mixture of microbial
tissue, lignin, cellulose, lipids, organic nitrogen compounds, and humic
"acid-like materials (McCoy, 1971).
At the present time there is a limited amount of data on the decomposition
of anaerobically digested sludge organics in soil. Thomas and Bendixen
(1969) studied the rate of decomposition of organic materials of septic
tank effluent and secondary effluent in sand and soil lysimeters. Data
from these studies indicated that these waste organics are readily bio-
degradable (67-89%) with little effect of temperature, loading rate, or
duration on the rate of degradation. The authors used their data to con-
clude that sewage sludges might be expected to degrade in a similar man-
ner and thus no detrimental accumulation of organic residues would occur.
It is doubtful, however, if this conclusion will be valid for anaerobically
digested sludges which have a much lower percent volatile solids than the
waste materials used by Thomas and Bendixen.
A recent report by ARS personnel, Beltsville, Maryland (1972) includes
studies on the biodegradation of a number of sludges including a digested
sludge. Less than 10 percent of the carbon from digested sludge added to
soil was evolved as C02 when incubated at 26ฐ for 54 days. At the same
time "raw" sludges showed an average loss of 27 percent carbon. These
studies would seem to provide data more in line with the expected bio-
logical stability of anaerobically digested sludges.
The studies in this section of the report were designed to provide add-
itional information on the rate of biodegradation of digested sludge and
to evaluate what influence soil properties, loading rate, soil moisture
content, and temperature would have on this microbial activity. In the
case of the temperature variable an attempt was made to duplicate natural
conditions by providing for both a diurnal as well as seasonal temperature
changes during the course of the study. Some recent reports have pointed
out that constant temperature studies of microbial activity in soils are
not useful in extrapolating laboratory data to actual field conditions.
One of the more significant results from this study was the observation
that the rate at which the organic carbon of digested sewage sludge was
evolved as C02 by microbial activity was largely independent of soil
properties. The obvious exception is soil moisture as influenced by
soil texture or soil structural features which will continue to have a
marked influence on water availability or soil aeration. These consider-
ations will be discussed in subsequent paragraphs. The data in Figures
2, 3, 4, and 5 are cumulative plots of C02 evolution from all three
17
-------
CO
2800
2400
2000
- 1600
o
UJ
o
I
CM
O
O
1200
800
400
' Temp ฐF
54.6-70.2ฐ I 43.2-66.3 133.6-51.3ฐ I 24.8-39.4ฐ I 22.2-37.8ฐ I 22.7-39.0ฐ
^224 Ton (metric)/ho
A
A Poulding cloy
o Celino silt loom
Ottokee sand
I I I 1 I I I I I I I I I I
20 40 60 80 100 120
Incubation Time (Days)
140 160 180
Figure 2. Cumulative plot of C02-C evolution from sludge amended soils during
temperatures equivalent to autumn-winter (Columbus, Ohio).
-------
2800
2400
2OOO
I 1600
o
o
1200
o
8~
800
400
Tempi ฐF
24.8-39.4ฐ 122.2-37.8ฐ I 22.7-39.0ฐ 131.2-49.9ฐ I 40.5-61.7ฐ I 50.3-72.8ฐ
A Poulding clay
o Celina silt loam
o Ottohee sand
224 Ton (metrii
80 80 100 120 140
Incubation Time (Days)
ISO 180
Figure 3. Cumulative plot of C02-C evolution from sludge amended soils during
temperatures equivalent to winter-spring (Columbus, Ohio).
-------
sludge amended soils over 6 month periods equivalent to autumn-winter,
winter-spring, spring- summer, and summer-autumn temperatures in Columbus,
Ohio. These data show that except for some unexplainable deviations by
the sludge amended Paulding clay (Figures 4 and 5) the correspondence
between soils with respect to CO, evolved was excellent. In practical
terms this means that at the rather high sludge loading rates employed
in this study (90 and 224 metric tons/ha) the initial soil properties
which would influence microbial activity are effectively masked, and
the sludge soil system is actually behaving as a sludge system. The
significance of this observation is that if soil moisture is neither
restrictive or excessive, anaerobically digested sewage added to low
fertility or marginal soils for renovative purposes could be expected
to decompose at the same rate as when applied to more fertile agri-
cultural soils.
The above data on the independence of sludge decomposition to soil
properties made it feasible to attempt to plot sludge decomposition as a
function of total heat imput to the soil-sludge system. The degree day
concept (see equation 1 below) was chosen as a useful indicator of heat
imput.
N f _ .
1) Monthly Degree Days = J ( XMT + X^ A ) x30
i=i / '
where XJCT = mean daily maximum temperature during a month (ฐF)
Xjnt = mean daily minimum temperature during a month (ฐF)
N = number of months
The relationship between degree days and % sludge carbon evolved as C0~
for the 90 and 224 metric ton amendments of sewage sludge is shown in
Figures 6 and 7. These data show quite clearly that a highly significant
correlation exists between C02 evolved and degree days after both one
and three month incubation periods. Correlation coefficients for data
obtained after 6 months incubation are not as high but still show a
statistically significant relationship (0001 probability) between tem-
perature and C02 evolution. Data of this type should make it possible
to predict the amount of decomposition of sewage sludge carbon per unit
time in different climatic regions using existing temperature data.
These studies also provide further evidence that anaerobically digested
sewage sludge is rather resistant to further biological decomposition
when added to soil. A. maximum of 20 percent of the added carbon was
evolved as COo from the 90 metric ton amendment (Figure 6) while slightly
less, 18 percent, was evolved from the larger, 224 metric ton amendment
(Figure 7) . The decreasing slope of the lines with time in Figures 6 and
7 also shows quite clearly that most of the sludge decomposition occurs
during the first months incubation. The obvious conclusion that can be
drawn from these data is that the addition of anaerobically digested
sewage sludge to soil will result in an increase in the level of soil
organic matter.
The previous discussion implied that the decomposition of anaerobically
digested sewage sludge was largely independent of differences in soil
20
-------
Tamp. ฐF
2800r31.2-49.9ฐ I 4Q5-6I.70 I 50.3-72.8ฐ I 59.3-81.6ฐ I 63.0-85.5ฐ I 61.8-838ฐ I
1 o
A O
2400
2000
1600
o
3>
|AJ
o 1200
CM
O
o
800
400
A Poulding clay
o Call no silt loam
O Ottofcoe sand
I i I I i i i i i i i i i
j i
20 40 60 80 100 120 140 160 180
Incubation Time (Days)
Figure 4. Cumulative plot of COo-C evolution from sludge amended soils during
temperatures equivalent to spring-summer (Columbus, Ohio).
-------
ro
to
2800
2400
2000-
- Temp.'F
59.3-81.6ฐ I 63.0-85.5ฐ I 61.8-83.8ฐ I 54.6-78.2 I 43.2-66.3ฐ 133.6-543ฐ
A Poulding cloy
o Celino silt loom
Ottokee sand
20 40 60 80 100 120
Incubation Time (Days)
I4O 160 180
Figure 5. Cumulative plot of CC>2-C evolution from sludge amended soils during
temperatures equivalent to summer-autumn (Columbus, Ohio).
-------
20
16
O
o
OT
0 12
o
s-
o
ฉ
0ป
o
(O
I mo.
3 mo.
90 Ton (metric)/ho
ฐ6 mo.
I I l l
2 4
Monthly
o
O Offokee sond
o Celino silt loom
A Poulding cloy
l l l l l
S
10
12
n I0~3
Figure 6. Relationship between % sludge carbon evolved as CO;, and degree dayss (Fฐ), (90
. metric ton/ha amendment).
-------
20
16
CM
O
o
w
O
-o 12
ฉ
o
o
&
CO
8
0
I mo
3 mo.
224 Ton (metric)/ho
6 mo.
II II I
Ottokee sand
o Celine silt loam
A Paulding clay
1_ 1 1 1
6 8
Degree Days, x 10"3
10
12
Figure 7. Relationship between 70 sludge carbon evolved as CC>2 and degree daysป(F )ป(224
metric ton/ha amendment).
-------
properties. One exception to this conclusion is the influence of soil
texture on the degree of microbial activity under 1/3 bar and saturated
moisture conditions. The influence of soil moisture on CO,, evolution
from sewage sludge amendments in the three soils for Experiment I is
shown in Figures 8, 9, 10. Note that the maximum microbial activity in
the Ottokee sand occurs under saturated moisture conditions at both the
90 and 224 metric ton loading rates (Figure 8). In the Celina silt loam
there is no difference in CO- evolution from soils at 1/3 bar and sat-
urated moisture conditions at the 224 metric ton additons, but a marked
reduction occurs under saturated conditions at the 90 metric ton rate
(Figure 9). With the Paulding clay saturated soil moisture conditons
severly restrict microbial activity at both loading rates (Figure 10).
The data provided above suggest that in the Ottokee sand sufficient 62
can diffuse into the soil columns at saturated moisture conditions to
maintain an active microbial population. The reduction of microbial
activity with the moisture content at 1/3 bar pressure is probably assoc-
iated with the high soluble salt content associated with the sludge
amendments which have increased the osmotic potential of the soil water
to a point where it was detrimental to microbial activity (See Phase 3S
Table 20). In the Paulding clay the effect of soil saturation in reducing
the diffusion of 02 into the soil columns resulting in anaerobic conditions
and reduced microbial activity is apparent. The same explanation is prob-
ably valid for the Celina silt loam soil at the 90 metric ton sludge
amendment. It is more difficult to explain the high microbial activity
in the Celina soil under saturated conditions at the high 224 metric
ton loading rate. Presumably the high osmotic potential associated with
soluble salts as well as possible effects associated with the increase
in organic matter have altered the activity of the water and allowed for
adequate aeration. In general, the effects of soil moisture shown in
Figures 8, 9, and 10 for Experiment I were consistent in all other experi-
ments. There were slight deviations, however, the most pronounced being
a more rapid decomposition of sewage sludge carbon in the Paulding soil
under saturated conditions when summer temperatures were maintained.
Tabular data on percent sludge carbon evolved as CO,, for all loading
ratess soils, moisture treatments and seasonal temperatures are shown in
Tables 7, 8 and 9.
These data point out the obvious importance of proper management of soils
to which liquid sewage sludge is applied. Saturation of fine textured
soils would certainly produce a slower rate of sludge decomposition and
associated problems of anaerobiosis and odor. Course textured sandy soils
would be less affected by saturated moisture conditions but might suffer
from periodic water deficits. Other problems associated with these soils
which will be discussed later e.g. downward movement of nitrate and other
soluble salts may limit their applicability as disposal sites.
25
-------
2400-
22.7ฐ-39ฐ
224 Ton-Sot
80 100 120
Incubation Time (Days)
180
Figure 8. Cumulative plot of CQ2=C evolution from sludge amended Ottokee sand
(autumn-winter temperatures, Columbus, Ohio).
Sat. = Soil saturated FซC. = Field capacity ie. moisture retained
in soil under 1/3 bar pressure.
-------
2400
2000
1600
1200
t.800
O
o
400
Temp ฐF
-54.6ฐ-78.2ฐ
43.2ฐ- 66.3ฐ
33.6ฐ-5l.3ฐ
24.8ฐ-39.4ฐ 22.2ฐ-37.8ฐ
224 Ton-Sat^
22.7ฐ-39.0ฐ,
20 40 60 80 100 120
Incubation Time (Days)
140 160
180
Figure 9. Cumulative plot of C02=C evolution from sludge amended Celina silt loam
(autumn-winter temperatures, Columbus, Ohio).
Sat. = Soil saturated F.Co = Field capacity ie. moisture retained in
soil under 1/3 bar pressure.
-------
ho
oo
Temp.ฐF
54.6-78.2ฐ
2400
24.8-39.4 22.2-37.8ฐ
20 40
SO 80 100 120
Incubofion Time (Days)
140 160 180
Figure 10. Cumulative plot of COg-C evolution from sludge amended Paulding clay
(autumn-winter temperatures, Columbus, Ohio),
Sat. = Soil saturated F.Co = Field capacity ie. moisture retained in
soil under 1/3 bar pressure.
-------
Table 7. SLUDGE DECOMPOSITION IN OTTOKEE SAND AS AFFECTED BY
SEASONAL TEMPERATURES
Temp.
Equiv.
Autumn
Winter
Exp. I
Winter
Spring
Exp. II
Spring
Summer
Exp. II I
Summer
Autumn
Exp. IV
Treatment3
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC.
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
% of Added Carbon Evolved as CO at.
1 Mo.
11.6
11.6
6.2
9.1 (9.6)b
0.9
1.5
0.5
0.8 (0.9)
4.4
5.0
3.1
3.5 (4.0)
11.9
11.0
8.1
9.4 (10.1)
3 Mo.
16.0
14.4
9.3
13.0 (13.1)
2.1
4.7
1.9
2.7 (2.9)
..12.5
12.6
11.1
11.9 (12.0)
17.1
16.1
15.0
15.4 (15.9)
6 Mo.
17.4
14.7
10.7
14.3
6.7
10.6
4.5
9.3
19.5
17.7
15.7
16.5
18.5
17.3
17.0
17.0
(14.3)
(7.8)
(17.4)
(17.5)
aSludge amendment in metric ton/ha. FC= field capacity
Sat. = saturated moisture conditions.
k Numbers in ( ) represent Mean % carbon evolved for each month
season combination.
29
-------
Table 8^ SLUDGE DECOMPOSITION IN CELINA SILT LOAM AS AFFECTED BY
SEASONAL TEMPERATURES
Temp.
Equiv.
Autumn
Winter
Exp. I
Winter
Spring
Exp. II
Spring
Summer
Exp. Ill
Summer
Autumn
Exp. IV
a
Treatment
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC.
90 Ton-Sat.
224 Ton-FC.
224 Ton-Sat.
% of Added Carbon Evolved as
1 Mo.
12.0
8.7
9.8
10.1 (10.1)
1.8
0.1
1.2
1.1 (1.1)
5.7
2.8
4.7
4.6 (4.5)
11.1
10.2
10.5
9.7 (10.4)
3 Mo.
17.7
14.6
13.8
14.3 (15.1)
4.4
1.0
3.6
3.2 (3.1)
13.3
9.8
12.2
11.7 (11.8)
19.7
17.0
15.4
15.7 (17.0)
CO- at.
6 Mo.
19.7
15.0
14.7
15.1
12.7
2.7
8.8
8.2
19.4
16.7
17.0
15.8
20.7
21.6
16.7
17.7'
(16.1)
(8.1)
(17.2)
(19.4)
a Sludge amendment in metric ton/ha. FC= field capacity
Sat. = saturated moisture conditions
b Numbers in ( ) represent Mean 70 carbon evolved for each month
season combination.
30
-------
Table 9. SLUDGE DECOMPOSITION IN :PAUH>ING CLAY AS AFFECTED BY
SEASONAL TEMPERATURES
Temp.
Equiv.
Au tumn
Winter
Exp.I
Winter
Spring
Exp. I
Spring
Summer
Exp. II I
Summer
Autumn
Exp. IV
a
Treatment
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
90 Ton-FC
90 Ton-Sat.
224 Ton-FC
224 Ton-Sat.
% of Added Carbon Evolved as
1 Mo.
12.9
1.1
10.2
1.8 (6.5)
2,7
0.3
1.2
0.3 (1.1)
4.4
0.1
4.0
0.4 (2.2)
14.3
3.0
9.6
8.6 (8.9)
3 Mo.
15.7
2.0
13.7
2.5 (8.5)
7.7
0.7
3.9
0.8 (3.3)
12.6
2.1
11.4
6.3 (8.1)
_
-
-
C02 at.
6 Mo.
16.5
2.0
14.9
3.0 (9.1)
15.1
5.3
9.4
2.9 (8.1)
.
-
-
_
-
-
a Sludge amendment in metric ton/ha. FC= field capacity
Sat= saturated moisture conditions
b Numbers in ( ) represent Mean % carbon evolved for each month
season combination.
31
-------
PHASE 2: " MICROBIOLOGICAL STUDY
One of the objectives of this study was an evaluation of the influence
on anaerobically digested sewage sludge on tnicrobial activity and
microbial population in soil. It was hoped that detailed studies of
the bacterial population would provide information to aid in solving
problems in public health, sewage sludge degradation, and the overall
management of soils amended with sewage sludge. Evidence for the
occurrence of specific physiological and morphological groups of
microorganisms should provide a reliable indicator of the success of
the disposal process and to forecast potential problems so that corrective
measures can be applied. The following paragraphs will provide data
which will show how well these objectives were met.
An overall evaluation of the activity of the heterotrophic microbial
population was obtained from the measurement of evolved C02-C from
sludge amended soils. Since the data and conclusions from this portion
of the study are given in Phase 1 of this report, no further discussion
will be provided in this section.
Numbers of aerobic heterotrophic bacteria and actinomycetes were estimated
by dilution plate counts on sludge-soil extract agar. No attempt was made
to separate and estimate the population of actinomycetes. Although one
must be aware of the obvious weakness of the plate count technique in
estimating the total population of bacteria in soil (Jensen, 1968), this
technique can provide useful relative information not attainable by any
other method.
Data on the numbers of bacteria and actinomycetes for all sludge loading
rates, soil moisture levels, and seasonal temperatures are shown for the
Ottokee sand (Figure 11), Celina silt loam (Figure 12), and Paulding clay
(Figure 13). A number of general conclusions can be drawn from these data:
1) Numbers of bacteria and actinomycetes are generally directly related
to the rate of sludge addition.
2) Maximum numbers of bacteria and actinomycetes were usually found
after one month's incubation and the numbers decreased after 3 and
6 month's incubation. The exceptions occurred in Experiment II,
where the incubation temperature during the first three months was
equivalent to winter temperatures in Columbus, Ohio. This trend in
population of bacteria and actinomycetes corresponds closely with
the C02 evolution data (Figures 2-5).
3) The largest numbers of bacteria and actinomycetes for soils incubated
at field capacity followed the order, Paulding clayปCelina silt
loamVOttokee sand. In saturated soils the order was reversed so
that numbers in the Ottokee sand>Celina silt loam> Paulding clay.
The latter trend was not consistent for the Paulding clay at the
high sludge loading rate. The high numbers of bacteria in the
Paulding clay at the 224 metric tons loading rate after three month's
incubation under saturated conditions in Experiments II and III
cannot be explained at this time.
32
-------
Bacteria 8 actinomycetes (x I0~6)/g soil
o> o ro A 2J
880888
l I
I I
o
01
I I
o>
o
o>
o
Figure Ho Dilution plate counts of bacteria and actinomycetes in sludge
amended Ottokee sand as influenced by loading rate, soil moisture
content^ time of incubation and temperature of incubation
(Experiments I-IV).,
-------
Bacteria and actinomycetes (x I0~6)/g soil
10
.p-
2r.
fO
o 8
en
1
-------
UJ
Ol
Bacteria 8 octinomycetes (x
Figure 130 Dilution plate counts of bacteria and actinomycetes in sludge amended Paulding
clay as influenced by loading rate, soil moisture content, time of incubation
and temperature of incubation (Experiments I-ปIV)o
-------
4) The effect of higher temperatures in increasing microbial activity
and numbers can be shown by the correspondence between numbers
of bacteria and actinomycetes and incubation temperature during.
the first months incubation for each experiment. Average incubation
temperatures followed the order Experiment I IV ? III ^11. For
the actual incubation temperatures see Table 2, page 7 .
Numbers of anaerobic or facultative anaerobic bacteria in the sludge
amended soils as determined by dilution plating are given in Figures 14,
15, 16. The following general conclusions can be.drawn from these data:
1) The largest population of anaerobic or faculatively anaerobic
bacteria was found in the Paulding clay and Celina silt loam
soils with a rather small number of anaerobic bacteria found in
the Ottokee sand. These results are expected, since the fine
textured soils would provide more opportunity for anaerobic micro-
sites than would a coarse textured soil such as the Ottokee.
2) The numbers of anaerobic or facultatively anaerobic bacteria
increased with increasing quantities of sewage sludge. These
data are as expected since large quantities of decomposable
s'ubstrate should reduce the oxygen tension in microsites so
that anaerobic bacteria could proliferate.
3) Also as expected, the numbers of anaerobic or facultatively
anaerobic bacteria generally increased in saturated soils where
oxygen diffusion would be restricted.
It is difficult to propose any significant role for the anaerobic bacteria
in the decomposition of sewage sludge in soil. Generally, the population
of anaerobic bacteria were less than 10 per cent of the total population
of aerobic heterotrophic bacteria. This was not true, however, in the
Celina silt loam soil amended with 90 metric tons of sludge in Experiment
IV (temperatures equivalent to summer-autumn). In this case the anaerobic
population approach 75-90 per cent of that determined under aerobic conditions.
It is highly probable that in this experiment, the preponderance of bacteria
growing under anaerobic conditions were faculative anaerobes.
Numbers of soil fungi were estimated by dilution plate counts on Rose Bengal-
Streptomycin agar. The weaknesses of this technique are even more severe for
soil fungi than bacteria because of the tendency of this technique to over-
estimate the profusely sporulating fungal species while drastically under-
estimating the sterile or slower growing species.
Data for the number of fungi in the Ottokee sand are shown in Figure 17,
the Celina silt loam in Figure 18, and in the Paulding clay in Figure 19.
The format for these Figures are the same as those discussed in the previous
sections. The following general conclusions can be drawn:
1) The fungal population increased in size in response to sludge
amendments. The direct relationship between the quantity of sludge
and numbers of fungi is not as pronounced as with bacteria and
actinomycetes.
36
-------
Anaerobic bacteria (ซ I0~s)/g soil
* fฐ <ฃ
o o o o
o o o o o
OJ
Figure lUป Dilution plate counts of anaerobic bacteria in sludge amended Ottokee sand
as influenced by loading rate, soil moisture content, time of incubation
and temperature of incubation (Experiments I-IV)ป
-------
Anaerobic bacteria (x
soil
oo
Figure 15. Dilution plate counts of aneerobic bacteria in sludge amended Celina silt
loam as influenced by loading rate, soil moisture content, time of in-
cubation and temperature of incubation (Experiments I-IV).
-------
Anaarobic bacteria (x IO"3 )/g soil
o
8
Figure 16. Dilution plate counts of anaerobic bacteria in sludge amended Paulding clay
as influenced by loading rate, soil moisture content, time of incubation and
temperature of incubation (Experiments I=IV).
-------
Fungi (ป
soil
Figure l?o Dilution plate counts of soil fungi in sludge amended Ottokee sand as
influenced by loading rate, soil moisture content, time of incubation
and temperature of incubation (Experiments I-IV),,
-------
8
Fungi (x KT^J/g soil
o o o o
OOP O O
3
p
at
3
o
a>
Figure 18. Dilution plate counts of soil fungi in sludge amended Celina silt loam as
influenced by loading rate, soil moisture content, time of incubation, and
temperature of incubation (Experiments I-IV).
-------
Fungi U I0"4)/g soil
Figure 19. Dilution plate counts of soil fungi in sludge amended Paulding clay as
influenced by loading rate, soil moisture content, time of incubation
and temperature of incubation.(Experiments I-IV).
-------
2) The number of fungi did not decrease substantially with time of
incubation as did the bacterial population. This might be expected
since numerous fungal species would sporulate during the initial
period of incubation and these spores would remain viable through
the three and six month incubation period.
3) Numbers of fungi were equal to or less than the control soils in
the sludge amended Paulding soil incubated at saturated moisture
conditions. This development of anaerobic conditions in the
saturated Paulding soil would eliminate fungi which are predomi-
nately aerobic.
4) In the Ottokee sand the largest numbers of fungi were also found
at field capacity. In this soil we find an inverse relationship
between the number of fungi and that of bacteria and actinomycetes
at both field capacity and saturation (See Figure 11). This same
relationship was not evident in either of the other two soils.
5) Numbers of fungi were less in the Celina silt loam incubated at
field capacity than in the other two soils. The population
difference between the soil incubated at field capacity and
saturated conditions was not as extreme, however.
A comparison of population changes of fungi and bacteria in response to
sludge amendments would indicate that both groups of organisms can participate
in sludge decomposition. Fungi are probably dominant in the decomposition
processes in the Ottokee sand at field capacity, while bacteria are dominant
under saturated moisture conditions. In the Paulding clay both populations
would seem to contribute significantly at field capacity, but the small
amount of sludge decomposition under saturated conditions is all bacterial.
In the Celina silt loam, bacteria probably dominate but fungi make a
significant contribution. Data on numbers of anaerobic bacteria are non-
conclusive and the significance of this group of microorganisms in sludge
decomposition cannot be evaluated.
Survival of Indicator Bacteria inSoil
Survival of pathogenic microorganisms in soils which are used for disposal
of waste effluents and sludges presents a potential health hazard which
could limit the applicability and success of this approach (Miller, 1973).
Surviving pathogens would provide a continuing risk to ground and surface
water supplies, possibly contaminate vegetation in the sludge amended site,
and present a health hazard to animals and humans in contact with the
contaminated soils or vegetation.
In this study the Membrane Filter technique (American Public Health Assoc.
et. al., Standard Methods, 1971) was used to evaluate the survival of total
coliforms, fecal coliforms, and fecal streptococci from the anaerobically
digested sewage sludge incorporated into soil. Data from these experiments
are summarized in Table 10. Only a relative evaluation of the survival of
these indicator bacteria are provided in Table 10 because variability between
duplicate columns at each sampling period was often very high. This high
43
-------
Table 10. RELATIVE SURVIVAL3 OF INDICATOR BACTERIA IN SOILS AMENDED WITH ANAEROBICALLY DIGESTED
SEWAGE SLUDGE.
Soil and
Treatment
Total Coliforms
1 mo. 3 mo. 6 mo.
Fecal Coliforms
Fecal Streptococci
1 mo.
3 mo.
6 mo.
1 mo.
3 mo.
6 mo.
Ottokee
90 Ton-FC
224 Ton-FC
ฑ
+
90 Ton-Sat.
224 Ton Sat.
+
^ Celina
ฃ 90 Ton-FC
224 Ton-FC
+
+
+
+
+
+
90 Ton-Sat.
224 Ton Sat.
+
+
+
+
Paulding
90 Ton-FC
224 Ton-FC
-ff-H-
90 Ton-Sat.
224 Ton-Sat.
+
+
a The relative comparisons above were based on the mean number of surviving indicator bacteria after
the indicated incubation time over all four experiments. The initial population of indicator bacteria
added with the sludge per gram of dry soil at the high and low loading rates was as follows: Total
coliforms, 1.1 x 10^ and 4.6 x W ; fecal coliforms, 1.2 x 10^ and 4.5 x 10^ ; fecal streptococci,
3.9 x 104 and 1.6 x 10^.
Relative survival % = -H-H-, 30-40%; -H-+, 10-307o,' ++, 1-10%; *t ฃ1.0%; ฑ, <0.1%; -, No bacteria detected.
-------
variability was associated primarily with the difficulty in predicting and
selecting what soil serial dilutions would provide the proper number of
colonies per filter. Over crowding or dilution to extinction were two
common problems which occurred. A. third problem was the interference of
clay and silt sized particles in the development of characteristic colonies.
It is the feeling of this investigator, however, that with greater experience
and more adequate replication, this technique can provide quantitative data
on survival of indicator bacteria.
A number of conclusions on the survival of indicator bacteria in the three
experimental soils can be drawn from the data in Table 10.
1) The population of all surviving indicator bacteria after 1 mnnths
incubation never exceeded 40 per cent of that originally added
with the sludge and continued to decrease with time.
2) In general, the percent survival of indicator bacteria was higher
in the Paulding clay than in the other two coarser textured soils.
This was particularly true for the fecal streptococci.
3) There was a trend for longer survival associated with soils
incubated under saturated moisture conditions.
4) The fecal coliforms, perhaps because of their lower initial
population,were almost completely eliminated from the soil by 3
month's incubation. Total coliforms and fecal streptococci were
often detected in low numbers after 6 month's incubation.
Characterjjga.tion of B acterial Isolates
Aerobic heterotrophic bacteria and actinomycetes were isolated from dilution
plates of sludge amended and control soil columns of all three experimental
soils after 1 month's incubation. Isolations were made from soil columns
incubated at each of the temperature series employed in .the study (Experiments
I-rV'). The techniques employed in the isolation and maintenance of the
isolates are described in the Materials and Methods.
A total of 354 bacterial isolates from the Ottokee sand and Celina silt loam
soils and an additional 67 isolates from 0 day control and sludge amended
treatments for these same soils were characterized extensively using the
morphological, cultural and biochemical characters listed in Table 6.
Characterization of bacterial isolates from the Ottokee sand and Celina silt
loam soils (Experiments III & IV) and the Paulding clay soil was limited to
morphological characters because of time limitations. In addition, none of
the actinomycete isolates have been characterized in detail at the present
time because of the inapplicability of many of the biochemical tests for
these organisms.
The total number of original isolates, the initial survival, subsequent
death and final number of bacterial isolates characterized are recorded
in Table 11 . The relatively low number of actinomyeetes isolated does
not indicate that they are less significant than the true bacteria in
45
-------
Table II, DISTRIBUTION OF BACTERIAL ISOLATES OBTAINED FROM DILUTION PLATING
Ottokee
Observation
Total No. of
Isolates
Bacteria
Actinomycetes
Yeasts
7o Loss at
Isolation
7o Subsequent
Loss
Field
Con-
trol
35
30
5
0
20.0
25.7
Capacity
90
Ton
40
38
2
0
27.5
5.0
224
Ton
31
31
0
0
25.8
9.7
Sand
Celina
Saturated
Con-
trol
36
33
3
0
36.0
11.1
90
Ton
40
40
0
0
47.5
5.0
224
Ton
40
40
0
0
12.5
2.5
Field Capacity
Con-
trol
40
35
5
0
30.0
15.0
90
Ton
48
56
2
0
10.3
6.9
224
Ton
61
59
2
0
24.6
9.8
Silt Loam
Saturated
Con-
trol
53
45
8
0
18.9
26.4
90
Ton
58
56
2
0
34.5
8.6
224
Ton
57
51
3
2
14.0
Mean
8.8
Mean
Totals
549
517
32
2
=25.1%
= 11.2%
Final No. of
Bacterial
Isolates
Characterized
19
27 20
19
19 34
22 48 40
29
33 44
354
-------
the decomposition of anaerobically digested sewage sludge in soil. Rather,
the numbers are low because isolations were made from plates at the highest
dilutions, which discriminated against actinomycetes which are less abundant
than the true bacteria. The same argument can be used to explain the low
incidence of yeasts among the isolates.
A mean value of 25.1 percent of the original isolates did not survive the
initial transfer and an additional 11.2 percent died in subsequent transfers
during the characterization period. Large but inconsistent differences in
survival were evident in the isolates from the various soils and soil
treatments (range of 10.3-47.5 percent loss). In general, the initial lack
of growth was more prevalent in the isolates from the Ottokee sand, but
death during subsequent transfers was higher in isolates from the Celina
silt loam. A slight trend toward a higher percentage of survival of the
isolates from sludge amended soils was also observed. The reason for poor
survival by some of the isolates has not been ascertained. Possible reasons
might include the choice of nutrient agar as the maintenance medium or the
choice of incubation and storage temperatures.
The characterization data for the bacterial isolates are shown in the
following tables. For convenience the results are listed in the following
subgroupings: Morphological and cultural characteristics of the completely
characterized isolated (Table 12) and morphological characteristics of
others (Tables 13 and 14); growth characteristics (Table 15); biochemical
and enzymatic activity (Table 16); acid production from selected carbohydrates,
aerobic (Table 17); acid production from selected carbohydrates, anaerobic
(Table 18); and antibiotic sensitivity (Table 19). The more significant
observations gleaned from these data tables will be discussed in the following
paragraphs. A more complete evaluation will require the development of
similarity matrices for the isolates using a system of Numerical Taxonomy.
The morphological and cultural characteristics of the bacterial isolates
from the isolates which were characterized in detail are summarized in
Table 12. It is important to note that moat generalizations which follow
in this and other paragraphs are consistent except for the Ottokee sand
treatments incubated at field capacity. Probably insufficient water was
present at field capacity in this coarse textured soil to provide water
films around soil particles as sites for bacterial proliferation. Reference
to Figure 11 supports this contention by showing that the bacterial
populations incteased very little in sludge amended Ottokee sand incubated
at field capacity.
Over 90 percent of the isolates from both soils and.in both the control
and sludge amended soils were rod or coccobacillary shaped cells.
Pleomorphic cells, curved rods, and cocci forms made up a small percentage
of the isolates and were found almost exclusively in the sludge amended
Celina soil. About 75 percent of the bacteria from sludge amended soils
occurred as single cells in culture, while those from the unamended soils
had a greater tendency to form chains of four or more cells. Of considerable
interest was the marked change of the bacterial population from one dominated
by gram positive bacteria in the .unamended soil to one where gram negative
bacteria constituted about 50 percent or greater of the population.
47
-------
Table 12. MORPHOLOGICAL AND CULTURAL CHARACTERISTICS OF BACTERIAL ISOLATES (EXPERIMENTS I & II)
-P-
oo
Ottokee Sand
Field Capacity
Character
Con-
trol
90
Ton
224
Ton
Saturated
Con-
rol
90
Ton
224
Ton
Celina Silt Loam
Field Capacity
Con-
trol
90
Ton
224
Ton
Saturated
Con-
rol
90
Ton
224
Ton
7. of Isolates Positive
Rods
Curved Rods
Cocco-
bacillary
Cocci
Spores
Pleomorphic
Singles
Chains
> 4 cells
Length-0.2-
0.6 IJLBJ
0 . 6 1.2 jim
"> 1.2 uia
Width-<0.5 \im
" 0.5-1.0 nm
">1.0 ^m
Gram Negative
" Positive
" Variable
Mobility
Colony White
" Yellow
" Pink
" Orange
" Purple
68.4
0.0
26.3
0.0
5.3
5.3
57.9
31.6
5.3
57.9
31.6
5.3
68.4
21.1
31.6
63.2
5.3
89.5
78.9
21.1
0.0
0.0
0.0
70.4
0.0
25.9
0.0
3.7
0.0
63.0
37.0
3.7
70.4
25.9
11.1
77.8
11.1
25.9
74.1
0.0
81.5
63.0
29.6
0.0
7.4
0.0
85.0
0.0
15.0
0.0
0.0
o.o
60.0
40.0
5.0
75.0
20.0
10.0
90.0
0.0
40.0
60.0
0.0
90.0
55.0
35.0
0.0
5.0
10.0
78.9
0.0
21.1
0.0
10.5
0.0
57.9
42.1
10.5
52.6
36.8
21.1
68.4
10.5
36.8
63.2
15.8
78.9
73.7
26.3
0,0
0.0
0.0
84.2
0.0
15.8
0.0
5.3
0.0
68.4
21.1
15.8
73.7
10.5
47.4
47.4
5.3
68.4
31.6
0.0
73.7
68.4
31.6
0.0
0.0
0.0
76.5
0.0
23.5
0.0
2.9
0.0
85.3
14.7
5.9
70.6
23.5
38.2
50.0
11.8
67.6
32.4
0.0
81.8
55.9
26.5
2.9
5.9
5.9
86.4
0.0
13.6
0.0
18.2
0.0
45.5
50.0
4.5
50.0
40.9
9.1
72.7
18.2
22.7
77.3
0.0
81.8
81.8
18.2
4.5
0,0
0.0
83.3
0.0
16.7
0.0
2.1
6.3
79.2
20.8
14.6
56.3
27.1
27.1
72.9
0*0
43.8
54.2
2.1
93.8
64.6
31.3
2.1
0.0
2.1
57.5
5.0
35.0
2.5
7.5
2.5.
75.0
22.5
20.0
57.5
,17.5
27.5
70.0
2.5
45.0
55.0
0.0
85.0
70.0
15.0
2.5
12.5
0.0
93.1
0.0
6.9
0.0
13.8
0.0
44.8
55.2
10.3
41.4
48.3
27.6
51.7
20.7
31.0
69.0
0.0
89.7
79.3
. 17.2
0.0
3.4
0.0
87.9
3.0
9.1
0.0
6.1
3.0
75.8
12.1
12.1
63.6
24.2
39.4
54.4
6.1
69.7
30.3
0.0
90.9
69.7
21.2
'0.0
0.0
9.1
88.6
0.0
9.1
2.3
2,3
0.0
79.5
20.5
15.9
52.3
31.8
27.3
63.6
9.1
52.3
47.7
0.0
79.5
56.8
34.1
0.0
2.3
6.8
-------
Table 13;. MORPHOLOGICAL -CHARACTERISTICS OF BACTERIAL ISOLATES FR6M THE OTTOKEE SAND AND CELINA SILT LOAM
SOILS (EXPERIMENTS III & IV).
Ottokee Sand
Field Capacity
Character
Con-
trol
90
Ton
224
Ton
Saturated
Con-
trol
90
Ton
224
Ton
Celina Silt Loam
Field Capacity
Con-
trol
90
Ton
224
Ton
Saturated
Con-
trol
90
Ton
224
Ton
% of Isolates Positive
Rods
Curved Rods
Cocco-
bacillary
Spores
Singles
Chains
>4 cells
Length
0.2-0.6 nin
" 0.6-1.2 urn
"> 1.2pm
t^idth
> 0.5 UK
" 0.5-1.0 Jim
" > 1.0 nm
Gram
Negative
" Positive
No. of
isolates
91.3
0.0
8.7
4.3
69.6
34.4
17.4
60.9
21.7
21.7
65.2
13.0
43.5
56.5
23
93.8
0.0
6.3
6.3
84.4
15.6
0.0
84.4
15.6
21.9
62.5
12.5
50oO
46o9
32
65.6
0.0
34.4
6.3
90.6
9.4
3.1
78.1
12.5
28.1
62.5
6.3
34.4
65.6
32
80.0
0.0
20.0
12.0
92.0
8.0
8.0
68.0
24.0
20.0
72.0
8.0
40.0
56.0
25
80.8
0.0
19.2
7.7
88.5
11.5
7.7
80.8
11.5
46.1
50.0
3.8
57.7
42,3
26
93.5
0.0
9.7
6.5
93.5
6.5
3.2
83.9
12.9
48.4
41.9
6.5
64.5
35 .5
31
78.3
4.3
34.8
17.4
82.6
21.1
8.7
73.9
34.8
13.0
95.7
8.7
17.4
82.6
23
86.7
0.0
13.3
13.3
60.0
40.0
6.7
46.7
46.7
13.3
46.7
40.0
20.0
80.0
15
71.4
0.0
26.7
14.3
85.7
14.3
0.0
78.6
21.4
28.6
57.1
14.3
35.7
64.3
14
86.7
0.0
13.3
6.7
80.0
20.0
0.0
66.7
33.3
13.3
73.3
13.3
26.7
73.3
15
93.8
0.0
6.3
6.3
87.5
6.3
12.5
81.3
6.3
31.3
68.8
0.0
56.3
43.8
16
83.3
0.0
16.7
0.0
94.4
5.6
0.0
88.8
16.7
22.2
77.8
0.0
50.0
50.0
18
-------
Table 14. MORPHOLOGICAL CHARACTERISTICS OF BACTERIAL ISOLATES FROM
THE PAULDING CLAY SOILS (EXPERIMENTS I - IV).
Paulding Clay
Field Capacity
Character
Rods
Cocco-
bacillary
Spores
Singles
Diplo
Form
Chains
? 4 cells
Length
0.2-0.6 nm
" 0.6-1.2 nm
" >1.2 urn
Width
> 0.5
" 0.5-1.0 jim
" ^1.0 M.HI
Gram
Negative
Positive
No. of
isolates
Con-
trol
100.0
0.0
8.3
66.7
0.0
33.3
0.0
66.7
33.3
0.0
75.0
25.0
25.0
75.0
12
90.
Ton
% of
100.0
0.0
0.0
69.2
0.0
30.8
23.0
38.5
38.5
38.5
38.5
23.0
46.2
53.8
13
224
Ton
Isolates
88.2
11.8
5.9
82.4
5.9
11.8
11.8
64.7
23.5
11.8
64.7
23.5
35.3
64.7
17
Con-
trol
Positive
78.6
21.4
7.1
71.4
0.0
28.6
0.0
64.3
35.7
7.1
64.3
28.6
7.1
92.9
14
Saturated
90
Ton
80.0
20.0
6.7
93.3
6.7
0.0
0,0
93.3
6.7
20.0
73.3
6.7
80.0
20.0
15
224
Ton
.66.7
40.0
0.0
66.7
6.7
20.0
13.3
60.0
26.7
0.0
73.3
26.7
73.3
26.7
15
50
-------
Table 15. GROWTH CHARACTERISTICS OF BACTERIAL ISOLATES
Ottokee Sand
Field Capacity
Character
Con-
trol .
90
Ton
224
Ton
Saturated
Con-
trol
90
Ton
224
Ton
Celina Silt Loam
Field Capacity
Con-
trol
90
Ton
224
Ton
Saturated
Con-
trol
90
Ton
224
Ton
70 of Isolates Positive
Relative
Growth3- 1
3
5
Even
Turbidity
Flocculent
Turbidity
Sediment
Pellicle
Growth at
" 20ฐC
" 35ฐC
Growth in
NaCl 2.57o
11 7.5%
" 12.57ป
77.8
11.1
11.1
6.3
6.3
87.5
0.0
42.1
89.5
68.4
94.7
36.8
5.3
65.4
19.2
15.4
19.0
0.0
71.4
9.5
63.0
96.3
70.4
96.3
40.7
7.4
80.0
10.0
10.0
5.3
0.0
57.9
26.3
70.0
100.0
45.0
95.0
25.0
5.0
78.9
10.5
10.5
10.5
0.0
89.5
0.0
47.4
94.7
89.5
94.7
15.8
0.0
63.2
26.3
10.5
21.1
0.0
73.7
5.3
57.9
100.0
68.4
73.4
21.1
5.3
33.3
25.0
37.5
29.4
0.0
58.8
11.8
76.5
94.1
67.6
88.2
26.5
5.9
95.5
4.5
0.0
11.8
5.9
82.4
0.0
59.1
100.0
81.8
72.7
27.3
4.5
64.6
20.8
12.5
10.4
0.0
85.4
4.2
68.8
95.5
77.1
93.8
45.8
6.3
40.0
53.3
6.7
20.0
5.0
67.5
7.5
55.0
95.0
77.5
97.5
60.0
12.5
63.0
29.6
3.7
10.3
0.0
75.9
10.3
24.7
93.1
82.8
86.2
24.1
0.0
41.9
38.7
12.9
40.6
5.0
37.5
15.6
51.5
100.0
69.7
75.8
33.3
9.1
44.8
24.1
31.0
29.5
0.0
52.3
18.2
52.3
97.7
72.7
93.2
27.3
2.3
Maximum growth in solution culture occurred after 1, 3 and 5 days of incubation.
-------
Table fc6. BIOCHEMICAL AND ฃNZฅMAIIC ACTIVITY-OF BACTERIAL ISOLATES
01
Ottokee
Field Capacity
Con-
Character trol
Hydrolysis of:
Starch 54 5
Cellulose 0.0
Pectin 4,5
Gelatin 28.6
Trybutyrin 0.0
Triolein 9.1
Production of t
Catalase 46.4
Cytochrome
Oxidase 27.3
Urease 22,7
Indole 0.0
Acetylmethyl-
Carbinol 0.0
Ac id (Methyl
red) 13 ซ6
H2S 0.0
Reduction of :
Nitrate (+gas) 9.1
Nitrate (no gas) 40.9
Utilization of
Citrate 13.6
Litmus Milk
Peptonized
Total 13.6
Surface 45.5
Acid 3.9
Colorless 22.7
Alkaline 36.4
Acid^Alkal. 22.7
Alkal.>Acid 13.6
90
Ton
20.8
0.0
5.0
27.1
2.1
3.3
58.3
31.3
14o6
0.0
0.0
0.0
0.0
6.3
37.5
16.0
22.9
33.3
18.2
0.0
45.8
35.4
20.8
224
Ton
15.0
0.0
12.5
17.5
0.0
7.5
67o5
47.5
17.5
2.6
5.1
7.7
0.0
, 7.7
33.3
12.5
10.0
32.5
14.6
10.0
32.5
30.0
12.5
Sand
Saturated
Con-
trol
31.0
0.0
6.9
24.1
0.0
10.3
44.8
27.6
13.8
3.6
0.0
10.3
0.0
6.9
48.3
10.3
13.8
51.7
10.0
3.4
48.3
27.6
34.5
90
Ton
27.3
0.0
6.1
30.3
6.1
9.1
54.5
63.6
9.1
3.0
0.0
3.0
0.0
9.1
45.5
39.4
33.3
33.3
17.2
9.1
51.5
27.3
27.3
224
Ton
% of
15.9
0.0
0.0
34.1
2.3
2.3
56.8
43.2
11.4
2.3
0.0
11.4
2.3
11.4
38.6
25.0
15.9
36.4
15.2
2.3
45.5
34.1
20.5"
Celina
Silt Loam
Field Capacity
Con-
trol
Isolates
31.6
0.0
5.3
31.6
0.0
OoO
57.9
26.3
15.8
0.0
5.3
5.6
0.0
5.3
0.0
5.3
26.3
21.1
21.1
10.5
10.5
21.1
5.3
90
Ton
Positive
22.2
0.0
0.0
18.5
3.7
0.0
88.9
18.5
13.3
0.0
3.7
3.7
0.0
11.1
40.7
37.0
7.4
55.6
3.7
22.2
48.1
25.9
3.7
224
Ton
0.0
0.0
0.0
15.0
0.0
0.0
80.0
55.0
15.0
0.0
0.0
0.0
0.0
10.0
25.0
25.0
5.0
30.0
0.0
10.0
40.0
30.0
10.0
Saturated
Con-
trol
42.1
0.0
5.3
21.1
0.0
15.8
42.1
31.6
26.3
0.0
0.0
10.5
0.0
5.3
21.1
26.3
10.5
26.3
21.1
5.3
26.3
36.8
0.0
90
Ton
10.5
0.0
0.0
15.8
5.3
0.0
78.9
36.8
26.3
0.0
5.3
5.6
0.0
5.3
42.1
47.4
15.8
15.8
10.5
0.0
47.4
26.3
15.8
224
Ton
2.9
0.0
6.7
8.8
0.0
20.6
58.8
50.0
11.8
0.0
0.0
0.0
3.1
3.1
43.8
51.5
8.8
23.5
10.5
5.9
47.1
11.8
17.6
-------
Table 17. 'ACID PRODUCTION FROM SELECTED CARBOHYDRATES (AEROBIC).
Ottokee Sand
Field Capacity
Carbohydrate
Glucose
Fructose
Mannose
Ga lactose
Arabinose
Lactose
Sucrose
Maltose
Cellobiose
Raff inose
Dextrin
Inulin
Mannitol
Sorb i to 1
Dulcitol
Ribose
Con-
trol
36.8
36.8
36.8
36.8
26.3
21.1
26.3
31.6
36.8
36.8
26.3
21.1
31.6
26.3
21.1
5.3
28.6
90
Ton
25.9
29.6
33.3
29.6
22.2
18.5
11.1
18.5
25.9
14.8
22.2
14.8
22.2
18.5
22.2
0.0
22.0
224
Ton
5.0
5.0
5.0
5.0
10.0
0.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
0.0
0.0
5.4
Saturated
Con-
trol
10.5
15.8
10.5
10.5
5.3
5.3
5.3
10.5
5.3
10.5
5.3
5.3
10.5
5.3
10.5
5.3
8.2
90
Ton
21.1
21.1
21.1
26.3
21.1
15.8
10.5
5.3
15.8
10.5
10.5
10.5
10.5
15.8
5.3
5.3
14.2
224
Ton
8.8
5.9
5.9
8.8
2.9
2.9
5.9
2.9
2.9
5.9
5.9
5.9
5.9
5.9
0.0
0.0
5.5
Celina Silt Loam
Field Capacity
Con-
trol
22.7
45.5
36.4
36.4
22.7
13.6
40.9
40.9
31.8
18.2
36.4
18.2
27.3
13.6
18.2
21.1
27.7
90
Ton
10.4
18.8
16.7
10.4
2.1
10.4
14.6
18.8
22.9
6.3
37.5
4.2
10.4
4.2
0.0
3.4
12.7
224
Ton
17.5
22.5
22.5
17.5
22.5
12.5
22.5
22.5
20.0
17.5
40.0
12.5
30.0
25.0
10.0
15.4
20.7
Saturated
Con-
trol
17.2
44.8
41.4
37.9
27.6
13.8
44.8
34.5
20.7
24.1
48.3
41.4
34.5
20.7
24.1
17.6
30.8
90
Ton
12.1
18.2
15.2
18.2
9.1
9.1
15.2
15.2
12.1
6.1
15.2
24.2
18.2
9.1
0.0
6.3
13.6
224
Ton
27.3
22.7
22.7
22.7
20.5
18.5
18.2
15.9
20.5
15.9
18.2
18.2
13.6
15.9
9.1
0.0
18.7
-------
Tab^e 18. ACID PRODUCTION FR6M SELECTED CARBOHYDRATES (ANAEROBIC)
01
p-
Ottokee Sand
Carbon
Sources
Field Capacity
Con-
trol
90
Ton
224
Ton
Saturated
Con-
trol
90
Ton
224
Ton
% of Isolates
Glucose
Fructose
Mannose
Galctose
Arabinose .
Lactose
Sucrose
Maltose
Cellobiose
Inulin
Sorbitol
Dulcitol
44.4
48.1
26.3
15.8
15.8
25.9
55.0
30.0
5.3
5.3
0.0
0.0
21.1
21.1
14.8
14.8
3.7
20.0
44.4
15.8
3.7
9.6
7.4
0.0
20.0
15.0
5.0
20.0
5.0
5.3
31.6
7.4
5.0
4.3
5.0
0.0
52.6
31.6
21.1
42.1
10.5
42.1
36.8
36.8
10.5
5.3
5.3
0.0
26.3
5.3
5.3
15.8
0.0
42.1
36.8
26.3
0.0
5.3
0.0
0.0
20.6
5.9
26.5
11.8
0.0
8.8
17.6
11.8
5.9
17.6
0.0
0.0
Celina Silt Loam
Field Capacity
Con-
trol
Positive
36.4
50.0
40.9
36.4
13.6
31.8
40.9
36.4
13.6
2-7.3
0.0
0.0
90
Ton
39.6
22.9
2.0.8
14.6
2.1
22.9
16.7
8.3
8.3
8.3
10.0
0.0
224
Ton
32.5
15.0
25.0
25.0
12.5
10.0
17.5
10.0
7.5
12.5
10.0
2.6
Saturated
Con-
trol
55.2
31.0
27.6
24.1
3.4
37.9
27.6
24.1
6.9
20.7
3.4
0.0
90
Ton
45.5
45.5
18.2
24.2
9.1
15.2
21.2
15.2
9.1
15.2
3.0
0.0
224
Ton
29.5
25.5
25.0
18.2
11.4
13.6
20.5
25.0
9.1
9.1
6.8
0.0
Mean
36.6
14.7
9.3
26.8 20.4 14.1
32.7 15.5 15.0
23.8 20.1 17.6
-------
Table-19. SENSITIVITY OF BACTERIAL ISOLATES, TO SEVEN SELECTED ANTIBIOTICS
Ottokee
Field Capacity
Antibiotic
Con-
trol
90
Ton
224
Ton
Sand
Celina Silt Loam
Saturated
Con-
trol
90
Ton
% Sensitive
Viomycin
Streptomycin
Chloromycetin
Novobiocin
Bacitracin
Tetracycline
Penicillin
63.2
89.5
89.5
94.7
68.4
100.0
68.4
70.4
88.9
88.9
77.8
77.8
96.3
63.0
50.0
95.0
90.0
75.0
65.0
90.0
70.0
68.4
84.2
78.9
63.2
52.6
94.7
52.6
47.4
73.7
89.5
63.2
52.6
100.0
42.1
224
Ton
Isolates
44.1
88.2
91.2
58.8
44.1
100.0
38.2
Field Capacity
Con-
trol
63.6
86.4
90.9
95.5
81.8
100.0
72.7
90
Ton
45.8
77.1
85.4
72.9
58.3
97.9
52.1
224
Ton
55.0
75.0
90.0
70.0
70.0
95.0
55.0
Saturated
Con-
trol
69.0
82.8
89.7
79.3
72.4
86.2
55.2
90
Ton
45.5
78\ 8
87.9
60.6
33.3
97.0
42.4
224
Ton
34.1
90.9
86.4
61.4
52.3
93.2
36.4
Mean 81.3 80.4 76.4 70.7 66.9 66.4 84.4 69.9 72.9 76.4 63.6 65.0
-------
This change and other changes discussed below reflects an alteration of
the soil microbial environment. The functional significance of these
changes is presently not apparent, but they are academically interesting.
Accompanying the change in gram reaction for bacterial isolates from
sludge amended soils was reduction in the number of spore formers, a
reduction in overall cell size and an increase in the number of pigmented
isolates. Most of the isolates were highly motile and did not differ
greatly regardless of their origin. This last result was opposite from
0 day isolates which average only 29 per cent motile. Morphological data
for the otherwise uncharacterized isolates from the Ottokee sand and Celina
silt loam (Table 13) and the Paulding clay (Table 14) agree closely with
the generalization above. In fact, the most dramatic change in population,
based on gram reaction, occurred in the Paulding clay soil columns incubated
under saturated conditions. Here the control population changed from
90 per cent gram positive bacteria to greater than 70 per cent gram
negative bacteria in the sludge amended treatments.
Bacterial isolates from sludge amended soils also differed considerably
in growth characteristics from the normal soil bacterial population '(Table 15),
Isolates from sludge amended soils usually grew at a faster relative growth
rate, grew better at 5* C but less well at 35* C, tolerated higher
concentrations of Na Cl, and exhibited a greater tendency for even turbidity
or pellicle formation in broth culture. The development of a more salt
tolerant population reflects the selection pressure associated with the
high accumulation of soluble salts in sludge amended soils.
Data on the biochemical activities of the bacterial isolates are provided
in Table 16. In general, the biochemical activities of the isolates did
not show the consistent trend differences between treatments previously
found with morphological and cultural characters. The exceptions to this
conclusion were the increased ability of isolates from sludge amended
soils to utilize citrate, to have increased catalase and cytochrome oxidase
activity, and reduced hydrolytic activity on starch. The ability to reduce
nitrate with and without production of Nฃ differed little in the Celina soil
isolates with and without sludge amendments, but was found more frequently
in isolates from sludge amended Ottokee sand. Proteolytic activity was
weaker in the bacterial isolates from sludge amended Ottokee sand (see data
for gelatin hydrolysis and peptonization of litmus milk) but not in the
Celina isolates. The biochemical tests frequently used for the character-
ization of enteric bacteria ie. production of ac ety line thy Icarbinol, acid
(methyl red), indol, and H^S, provided a very low percentage of positive
responses with no apparent differences between soils or treatments. Lipid
esterase, cellulase, and pectinase activity were either low or entirely
absent among the isolates. The combination of responses possible on litmus
milk were largely inconsistent.
The ability of the bacterial isolates to produce acid from a series of
selected carbohydrates was evaluated under both aerobic and anaerobic
conditions (Tables 17 and 18). Bacterial isolates from sludge amended
soils were less able to produce acid from the various carbohydrates under
aerobic conditions than were the isolates from the control soils. The
overall activity of the isolates from the saturated Celina soil also surpassed
that of the saturated Ottokee sand isolates. The same general trends
were evident under anaerobic conditions but the overall number of positive
cultures from the Ottokee sand increased considerably. Antibiotic
56
-------
FIELD CAPACITY
SATURATED
o
en
D 224 Ton
O 90 Ton
Control
601 3
Incubation Timo (Months)
Figure 20. Soil pH in incubated columns of sludge amended
and control Ottokee sand (Experiments I-IV).
57
-------
FIELD CAPACITY
SATURATED
O 224 Ton
o 90 Ton
Control
601 3
Incubation Time (Months)
Figure 21. Soil pH in incubated columns of sludge amended
and control Celina silt loam (Experiments I-IV)
58
-------
FIELD CAPACITY
SATURATED
0224 Ton
090 Ton
ฎ Control
601 3
Incubation Timo (Months)
Figure 22. Soil pH in incubated columns of sludge amended
and control Paulding clay (Experiments I-IV).
59
-------
9.0
8.0
7.0
6.0
. Field Capacity Saturated
,:, Ottokee
Solution
Control
Control
9.0 r
8.0
i
a.
7.0
90 Ton
*--.
""""^^^
e
6.0
9.0 r
8.0
7.0
90 Ton
6.0
224 Ton
224 Ton
0 I
601 3
Time (months)
i
6
Figure 23. Relationship of solution pH to soil pH (Ottokee
sand).
60
-------
X
O.
9.0
8.0
7.0
6.0
9.0
8.0
7.0
6.0 LL-
9.0
Field Copocity
Soil
Solution
Celino
Saturated
Control
Control
90 Ton
90 Ton
8.0
7.0
60
224 Ton
224 Ton
0 I
60 I
Time (months)
6
Figure 24. Relationship of solution pH to soil pH (Celina
silt loam)
61
-------
85
7.5
6.5
5.5
Saturated
Soil
Solution
Control
Paulding
Control
t 1 1
i
a.
8.5 r -
7.5
6.5
5.5
90 Ton
90 Ton
8.5 r
7.5
6.5
5.5
224 Ton
224 Ton
0 I
601
Time (months)
Figure 25. Relationship of solution pH to soil pH (Paulding
clay).
62
-------
sensitivity was the last group of characters utilized for the evaluation
of the bacterial isolates. The data shown in Table 19 shows a general
increase in resistance to four of the seven antibiotics. The exceptions
were streptomycin, chloromycetin and tetracycline. As was the case with
a number of other characters, the isolates from the sludge amended Ottokee
sand treatments incubated at field capacity differed little in antibiotic
sensitivity from the control.
PHASE 3: ANALYSES OF THE DISPLACED SOIL SOLUTIONS
Analyses of the displaced soil solutions from the various sludge amended
columns provide an indirect evaluation of microbial activity. The presence
or absence of Various inorganic or organic compounds or ions in the soil
solution eg. soluble salts, nitrogen compounds, heavy metal ions, etc. are
often directly or indirectly related to microbial activity. The presence
of these soluble materials is of considerable importance to the utility of
sludge disposal on soil, since excessively large accumulations of some ions
or organic compounds could affect ground water quality if movement through
the profile would occur or be phytotoxic to the associated vegetative cover.
Since these studies were based on laboratory incubation experiments, the
concentration of soluble constituents found may not be directly applicable
to field conditions. Under natural conditions both plant uptake and leaching
could be expected to decrease the concentration of soluble compounds found
at any one time in the soil plow layer. However, it should be pointed out
that the concentrations of any one soluble constituent found in this study
will reflect the total supplying power of the incorporated sewage sludge
for this constituent during a particular set of experimental conditions.
Soil and Solution pH
Hydrogen ion activity in the sludge amended soil is of considerable
significance because of the impact of pH on both microbial activity and
the solubility and availability of many nutrient and heavy metal cations.
Data showing changes in the soil pH with time under different temperature
of incubation are shown in Figures 20, 21, 22. In general, the addition of
anaerobically digested sewage sludge raised the initial pH of all three
soils. This was followed by a subsequent drop in pH in all soil treatments
where appreciable nitrification of sludge nitrogen occurred. (See Figures
26-43). The magnitude of this pH drop was almost always directly related
to the amount of nitrate formed. The pH of sludge amended soils remained
above the control soils in those treatments where anaerobic conditions
decreased nitrification or accelerated biological denitrification, or where
an apparent sludge toxicity decreased the activity of the nitrifying
bacteria. These instances will be noted specifically in the subsequent
discussion of soluble nitrogen.
What does seem readily apparent is that the addition of sewage sludge to
soils maintained under proper moisture conditions will result in an appreciable
decrease in soil pH. This drop in pH could alter the solubility of various
metal cations, that this actually occurred can be seen from the plant analysis
data shown in Figures 49 & 50 for Cu and Zn and perhaps for B (Figure 52).
63
-------
The relationship of the pH of the displaced soil solution and the soil pH
is shown in Figures 23, 24, 25. In general, the solution pH was considerably
higher than that of the soil from which it was displaced, the magnitude of
the difference being greater in the finer textured Paulding and Celina soils.
These differences can be explained as an example of the suspension effect
(McLean and Franklin, 1964) which increases the activity of cations near the
surface of the soil colloids (in this case IT1") compared to the displaced
soil solution. The observations that the difference in hydrogen ion activity
between the soil and displaced soil solution decreases as the electrolyte
concentration increases eg. higher rates of sludge amendment, or when the
soils are incubated at field capacity rather than under saturated moisture
conditions, are also in line with the idea of a suspension effect (Franklin
and McLean, 1963).
Specific Conductance
The accumulation of excess soluble salts after application of sewage sludge
to soil is another potential problem which might affect the utility of land
disposal of sludges (Hinesly et.al. 1972). Plant growth and seed germination
are both adversely affected by soluble salt accumulation, although plant
species differ greatly in their tolerance (Richards, 1954). The mechanisms
responsible for reduced growth include the reduced availability of soil
water (osmotic effects), toxicity of certain of the soluble ions, and the
induction of nutrient imbalances because of excess salts.
Specific conductance of the displaced soil solutions from sludge amended
and control soils was measured to evaluate the possible severity of salt
accumulations. These data are shown in Tables 20, 21, 22. The relationship
of specific conductivity to a potential crop response can be estimated from
the response table for saturation extracts shown below (Table 23).
Table 23. RELATIONSHIP OF CROP RESPONSE TO SOIL SALINITY EXPRESSED IN
TERMS OF THE CONDUCTIVITY OF THE SATURATION EXTRACT. (RICHARDS, 1954)
Saline
effects
nostly
negligible
Yields of
very sen-
sitive crops
may be re-
stricted
Yields of
many crops
restricted
0 2 4 j
Only tolerant
crops yield
satisfactorily
Only a few
very tolerant
crops yield
satisfactorily
3 16
Scale of conductivity (mmhos/cm at 25 C)
Specific conductivity of displaced soil solutions from soils incubated under
saturated soil conditions may be compared directly with Table 23. Specific
conductivity of solutions from soils incubated at field capacity should be
reduced about 1/3 in the Celina and Paulding soil and by 1/2 in the Ottokee
sand for direct comparison with the saturation extract.
It is evident from Table 20 that accumulation of soluble salts could be a
64
-------
Table 20. SPECIFIC CONDUCTANCE OF SOIL SOLUTIONS DISPLACED FROM SLUDGE
AMENDED OTTOKEE SAND.3 VALUES ARE MEANS OF DUPLICATE COLUMNS.
Exp. Incubation Specific Conductance (mmho/cm)^
No. Time Field Capacity Saturated
(mo.) Control 90 Ton 224 Ton Control 90 Ton 224 Ton
I 1 1.2(0.4) 11.1(3.3) 10.4(3.1) 0.6(0.2) 6.7(2.0) 12.5(3.8)
3 1.3(0.4) 11.8(3.5.) 13.6(4.1) 0.8(0.2) 8.2(2.5) 15.0(4.5)
6 1.7(0.5) 16.0(4.8) 18.0(5.4) 1.0(0.3) 10.3(3.1)15.5(4.7)
II 1 0.9(0.3) 9.0(2.7) 9.5(2.9) 0.4(0.1) 1.5(0.5) 8.7(2.6)
3 1.0(0.3) 11.3(3.4) 16.0(4.8) 0.8(0.2) 6.5(2.0) 13.3(4.0)
6 1.6(0.5) 12.9(3.9)20.2(6.1) 1.0(0.3) 9.5(2.9) 16.3(4.9)
III 1 1.0(0.3) 10.5(3.2)13.5(4.1) 0.9(0.3) 6.4(1.9) 12.5(3.8)
3 2.2(0.7) 11.9(3.6)16.9(5.1) 0.8(0.2) 10.5(3.2) 17.0(5.1)
6 3.4(1.0) 21.3(6.4)16.5(5.0) 1.4(0.4) 13.2(4.0) 18.3(5.5)
IV 1 1.1(0.3) 11.1(3.3)16.8(5.0) 0.6(0.2) 7.5(2.3) 12.5(3.8)
3 2.0(0.6) 19.0(5.7)21.8(6.5) 1.1-(0.3) 9.5(2.9) 15.3(4.6)
6 2.6(0.8) 17.8(5.3)19.8(5.9) 1.4(0.4) 10.8(3.2) 17.8(5.3)
0 day 0.5(0.2) 3.3(1.0) 7.7(2.3) 0.3(0.1) 2.0(0.6) 5.2(1.6)
a Sludge amendments in metric tons/ha
''Values in ( ) are calculated osmotic pressures (atm.) using a factor
of 0.3 X Specific Conductance (mmho/cm). (Jackson, 1958)
65
-------
Table 21. SPECIFIC CONDUCTANCE OF SOIL SOLUTIONS DISPLACED FROM
SLUDGE AMENDED GELINA SILT LOAM.a VALUES ARE MEANS OF DUPLICATE
COLUMNS.
Exp. Incubation Specific Conductance (mmho/cm)
No. Time Field Capacity Saturated
(mo.) Control 90 Ton 224 Ton Control 90 Ton 224 Ton
I 1 1.9(0.6) 9.3(2.8) 9.5(2.9)1.5(0.5) 6.9(2.1). 6.9(2.1)
3 2.1(0.6) 11.3(3.4) 15.3(4.6) 1.0(0.3) 4.3(1.3) 7.5(2.3)
6 2.8(0.8) 11.1(3.3) 14.4(4.3) 1.3(0.4) 4.3(1.3)16.5(5.0)
II 1 2.4(0.7) 4.8(1.4) 7.2(2.2)2.5(0.8) 4.1(1.2)5.7(1.7)
3 2.5(0.8) 5.0(1.5) 8.4(2.5) 2.0(0.6) 3.2(1.0) 5.9(1.8)
6 3.2(1.0) 13.0(4.5) 10.1(3.0) 1.9(0.6) 3.0(0.9)13.4(4.0)
III 1 2.3(0.7) 6.0(1.8) 9.1(2.7) 1.8(0.5) 3.3(1.0)10.3(3.1)
3 2.8(0.8) 12.6(3.8) 14.5(4.4) 1,4(0.4) 4.7(1.4)14.3(4.3)
6 3.8(1.1) 17.8(5.3) 21.8(6.5) 1.3(0.4) 4.3(1.3)18.8(5.6)'
IV 1 2.1(0.6) 8.0(2.4) 10.8(3.2) 2.5(0.8) 5.0(1.5)12.5(3.8)
3 3.2(1.0) 13.1(3.9) 16.4(4.9) 1.3(0.4) 4.4(1.3)12.2(3.7)
6 3.9(1.2) 16.3(4.9) 21.5(6.5) 1.2(0.4) 4.1(1.2)14.2(4.3)
0 day 1.8(0.5) 3.3(1.0) 6.3(1.9) 1.5(0.5) 3.3(1.0)6.0(1.8)
a Sludge amendments in metric tons/ha,
b Values in ( ) are calculated osmotic pressures (atm.) using a
factor of 0.3 X Specific Conductance (mmho/cm). (Jackson, 1958)
66
-------
Table 22. SPECIFIC CONDUCTANCE OF SOIL SOLUTIONS DISPLACED FROM SLUDGE
AMENDED PAULDING CLAY.3 VALUES ARE MEANS OF DUPLICATE COLUMNS.
Exp. Incubation Specific Conductance (mmho/cm)"
No. Time Field Capacity Saturated
(mo.) Control 90 Ton 224 Ton Control 90 Ton 224 Ton
I 1 1.4(0.4) 1.8(0.5) 1.6(0.5) 0.8(0.2) 1.2(0.4) 2.0(0.6)
3 0.4(0.1) 0.5(0.2) 1.8(0.5) 0.5(0.2) ' 1.8(0.5) 2.9(0.9)
6 0.3(0.1) 0.6(0.2) 1.8(0.5) 1.0(0.3) 2.7(0.8) 3.2(1.0)
II 1 0.4(0.1) 0.3(0.1) 1.3(0.4) 0.6(0.2) 0.9(0.3) 1.8(0.5)
3 0.7(0.2) 2.4(0.7) 1,9(0.6) 0.9(0.3) 2.1(0.6) 2.3(0.7)
6 0.3(0.1) 0.5(0.2) 1.8(0.5) 0.3(0.1) 1.5(0.5) 1.8(0.5)
III 1 1.1(0.3) 1.0(0.3) 1.6(0.5) 1.0(0.3) 0.9(0.3) 1.6(0.5)
3 1.1(0.5) 1.1(0.3) 0.8(0.2) 1.2(0.4) 0.9(0.3) 1.5(0.5)
6
IV 1 0.9(0.3) 0.7(0.2) 0.9(0.3) 1.1(0.3) 0.7(0.2) 1.7(0.5)
3 __ .. ._ _ .. .
6
0 day 0.5(0.2) 0.7(0.2) 2.1(0.6) 0.9(0.3) 1.0(0.3) 2.5(0.8)
3 Sludge amendments in metric tons/ha
b Values in ( ) are calculated osmotic pressures (atm.) using a factor
of 0.3 X Specific Conductance (mmho/cm). (Jackson, 1958)
67
-------
potentially severe problem in the Ottokee sand at both sludge loading rates |
and at both soil moisture contents. A severe problem could also exist in |
the Celina soil at both loading rates at field capacity, but only at the !
high 224 metric ton loading rate under saturated conditions (Table 21).
In contrast the accumulation of salts in the soil solution of the Paulding
clay would have minimal effects on plant growth (Table 22). It should also I
be noted that specific conductivity of soil solutions increased with time |
of incubation and w: th increases in incubation temperature. Both observations i
point to the significance of the microbial population in mineralization
reactions which contribute to soluble salts. Certainly high concentrations
of soluble mineral nitrogen have made an appreciable contribution to the
specific conductivity in the Ottokee sand and Celina silt loam.
The question might be asked, "How well can these laboratory incubation
data be extrapolated to field conditions where rainfall and plant uptake
would be expected to reduce accumulation of soluble salts?" As discussed
previously, these data would provide information on the potential quantity
of soluble salts which could be released to the surrounding soil environment
during a set period of incubation. Under conditions of minimal precipitation,
or in soils with an impermeable B horizon, plowsole or pan, leaching of
salts from the surface soil would not occur, and the potential for salt
accumulation would exist. Hinesly et al. (1972) have speculated that salt
accumulation may have adversely affected corn yields on sludge treated plots
during the dry conditions experienced in 1971.
Organic Matter
Soluble organic matter in soils amended with high rates of sewage sludge
could be of significance as a potential pollutant of ground or surface
water supplies. In addition many soluble organic compounds are capable
of complexing and solubilizing heavy metal ions which would alter the. plant
availability and downward movement of these ions in the soil.
The organic matter content of the displaced soil solutions from all three
soils averaged for all four temperature cycles are shown in Table 24. High
concentrations of soluble organic matter were present in the soil solution
from the sludge amended Ottokee sand incubated at field capacity. The amount
of organic matter was directly related to the rate of sludge amendment.
Moderate levels of soluble organic matter were present in the Ottokee
sand under saturated conditions, and in the Celina silt loam soil at both
moisture contents. The concentration of soluble organic matter in the
Paulding clay was 10 to 30 times lower than that in the Ottokee sand and
increased only slightly above the unamended control soils. Also note
that the soluble organic matter in sludge amended soils arises primarily
from the sludge itself (see 0-day concentrations of soluble organic matter)
and decreases slightly with incubation. The one exception to this is in
the Ottokee sand, incubated at field capacity. Here an increase in soluble
organic matter occurred with incubation.
These data suggest that soluble organic matter could present a pollution
hazard to ground water in sludge amended coarse textured soils like the
Ottokee. Accumulation of soluble organic matter should not be a pollution
68
-------
Table 24-. ORGANIC MATTER CONTENT OF SOIL SOLUTIONS DISPLACED FROM SLUDGE AMENDE-D SOILS.
VO
Organic Matter Content (mg/ml^
Soil
Ottokee
Sand
Celina
Silt
Loam
Paulding
Clay
Incubation
Time
0 day
1 mo.
3 mo.
6 mo.
0 day
1 mo.
3 mo-
6 mo.
0 day
1 mo.
3 mo.
6 mo0
Field Capacity
Control
0.27
0.29
0.34
0.40
0.35
0.53
0.43
0.49
0.17
0.41
0.29
0.13
90 Ton
4.6
26.5
48.9
33.5
6.3
3.7
4.5
3.5
0.35
0.36
0.21
0.16
224 Ton
27.8
93.6
95.0
104.3
9.8
7.8
10.4
8.8
0.58
0.54
0.66
0.50
Control
0.24
0.26
0.34
0.27
0.25
0.54
0.54
0.54
0.16
0.33
0.25
0.15
Saturated
90 Ton
4.3
3.6
3.6
2.2
4.4
3,2
4.3
3.5
0.20
0.28
0.27
0.17
224 Ton
8.3
8.4
7.3
7.9
7.8
5.9
6.6
5.5
0.47
0.45
0.34
0.35
aValues given are the means for displaced soil solutions from duplicate columns for
Experiments I-IV. . '
-------
hazzard in finer textured soils such as the Celina or Paulding where
adsorption of organic matter on soil colloids would prevent appreciable
downward leaching. The concentration of soluble organic matter found in
the soil solution of the Celina silt loam could, however, significantly
influence the solubility and mobility of heavy metal ions.
Nitrogen Transformations
Nitrogen reactions in sludge amended soil are of great significance to
the success of land disposal of sewage sludges. Accumulation of nitrate
in the soil at concentrations greater than that which can be utilized by
the associated crop or vegetation, could result in nitrate movement into
ground water. Hinsely et.al. (1972) have proposed that excessive formation
of nitrate from sewage sludge nitrogen is the factor most likely to
determine the maximum rate of sludge additions to soils.
In this reporti data are presented on the distribution of organic, NHA ,
N02 ", and NC>3 nitrogen in the displaced soil solution from the
Ottokee sand and Celina silt loam; and for NHฃ and N03~ nitrogen from
the Paulding clay. In the latter soil preliminary analysis of the soil
solutions showed both NOo and organic nitrogen to be absent or present
in negligible quantities. Nitrogen data reported in Figures 26 to 43 show
the differences in soil nitrogen associated with the differing sludge
loading rates, soil moisture contents, and temperatures of incubation
(Expt. I to IV). Although these data are reported in ug Nfail of the soil
solution, a conversion to ug/g dry soil can be made by multiplying the
following conversion factors.
Ottokee sand -field capacity 0.080
-saturated 0.200
Celina silt loam -field capacity 0.215
-saturated 0.350
Paulding clay -field capacity 0.329
-saturated 0.620
Each soil exhibited characteristic soluble nitrogen accumulation curves.
For example, ammonium nitrogen and soluble organic nitrogen were the most
significant forms of nitrogen found in sludge amended Ottokee sand after
1 month's incubation. It seems obvious from these data that some component
of sewage sludge inhibited nitrification while allowing aramonification to
proceed. The duration of this inhibition of nitrification varied and was
more prolonged at the higher sludge loading rates, and at the cooler
incubation temperatures. As would be expected the concentration of ammonium
nitrogen during subsequent incubation was inversely related to nitrate
accumulation. This was1also true to a certain degree with soluble organic
nitrogen. There was no absolute evidence of denitrification in the Ottokee
sand although the leveling off of the rate of accumulation of nitrate during
the last three month's incubation could suggest at least some loss of nitrate
through denitrification.
70
-------
3000
~-
0-2000
*~*^
c
0
b.
.^
Z
1000
(
Q
224 Metric Ton
-
/
/
/
y
t
/
/
/
S
'
/
?
f
- 1
1
ป >t**
* t,'*
<
.X
**"* *""" ^3
"> JIM /'' .L i r"J^ T
90 Metric Ton
^^"""'0
*** *ฐ^^ x"
p*"" /
1 /
1 /
1 .'
I S
1 /
'>*?,.:.. ^
D 1 3 601 3 6
Control
-
Org. -N
MLJ + _ft| .....
IMrl^ IM ~
N03~ -N
N0g~ -N
-
o
X
x'
x'
x'
_x
^-"'ฐ
ฎ
/
/
7
01" 3 6
300
-.
ฃ
*x
O>
200 3
c
a>
o>
o
^
Z
100
Incubation Time (months)
Figure 26. Soluble nitrogen in displaced soil solutions from sludge amended and
control Ottokee sand incubated at field capacity (Experiment I, autumn-
winter) .
-------
3000
E
x
o>
c
I
1000
224 Metric Ton
0 I
90 Metric Ton
Control
Org. -N
NH4^ -N T-
N03~ -N
N02~ -N
300
200
c
o
100
60 13 60 I
Incubation Time (months)
Figure 27. Soluble nitrogen in displaced soil solutions from sludge amended and
control Ottdkee sand incubated under saturated conditions (Experiment I,
autumn-winter).
-------
3000
**^^
ฃ
X.
o>
32000
c
Ol
oซ
o
1000
224 Metric Ton
r
i
i
/
t
t
/
i
i
t
i
j
/
t
/
/ 0
,' P :..
"\
'':. i
[TTTTTW^TBrirTrnriyi TFJ^^*^^yป^**^ป,
90 Metric Ton
/--._
/
/ ^B
* /
/..ป.., y'
'' ""*;ปป;
1 i -.-A* 1.4
Control
-
Org. -N -
NH4* -N
j^Q ~ ^ ......
NOjf -N
-
/
/
/
/'
* - ^ ''
/ '--^
f-
300
^
E
o>
200^
C
O*
o
w.
100
0 I
SO I 3 60 I
Incubation Time (months)
Figure 28. Soluble nitrogen in displaced soil solutions from sludge amended and con-
trol Ottokee sand incubated at field capacity (Experiment II, winter-
spring) .
-------
3000
0>
^2000
o<
o
1000
224 Metric Ton
0 I
90 Metric Ton
Control
Org. -N
NH4+-N-
N03~ -N
N02~ -N-
601 3 601
Incubation Time (months)
300
E
x
01
20O ~
100
Figure 29. Soluble nitrogen in displaced soil solutions from sludge amended and con-
trol Ottokee sand incubated under saturated conditions (Experiment II,
winter-spring).
-------
-4
Ln
3000
E
X
o<
~1
- 2000
c
OJ
CP
o
~
z
1000
224 Metric Ton
/ ~~~~~- -0
/
/
/
1
1
!
'
/
/
?
/
/
/
-/
. q
/
/ '*
f /
'.^
i; _ r^fa.^Z^.A
90 Metric Ton ,ฐ
/
/
/
/
/
/
/
/
/
/
/
/
x^^ 'Xx
?* / X
/ v
/ / >
/ /
/ /
*?....
Control
Org. -N - /
NH4* -N /
H /
N03~ -N /
N02- -M f
/
/
j
:
9
:
f
.'
j
!
f
!
L'f'~~~.^-~>f~ _-... i
300
-.
?
o>
200 -
^
41
O>
0
Z
100
0 I
60 I 3 60 I
Incubotion Time (months)
Figure 30. Soluble nitrogen in displaced soil solutions from sludge amended and con-
trol Ottokee sand incubated at field capacity (Experiment III, spring-
summer) .
-------
3000 -
-T2000 -
c
1>
1000
224 Metric Ton
-
/
j
j
/' \ '
X ^ /
/ \ j
/r >
' \
y \
/ t \
. 1 ; \
1 /
1
1 /
1 i
..*.. !
-'" \.J
___tปjj' ' '* ij.'. i J.
90 Metric Ton
."
fS
s
X
x'
x
/
/
/
/
- /
/ ><^
/ / ^^^^^
- '' , i f "" " i.r . 1
Control
-
Org. -N
NH4+-N
N03- -N
N02- -N
-
s*
s'
S' '
s'
s'
Jt
.s'
f.'
''*-
p-*l^l ~~~--ฃ _ 1
300
-^
E
x
200 5
c
V
o
-
z
100
60 I 3 60 I 3
Incubation Time (months) ,
Figure 31. Soluble nitrogen in displaced soil solutions from sludge amended and con-
trol Ottokee sand incubated under saturated conditions (Experiment III,
spring-summer).
-------
3000
52000
c
0>
o>
o
1000
224 Metric Ton
/ ^ o
1 >'
1 x- -o
- 1 .''
i S'
1 '
1 i
1 i
1 !
I i
\ j
I
. !
' ..ฎ../.
i..--ฐฐ / *
/
.' . . . ซ
D 1 3 6<
90 Metric Ton
r -ซ
/
/
/
/
j
j
i
*
1 i ^
/ / X--
/ "^^
/ . i
"":i^ -j .......ฑ
313 6(
Control
."*
i
j
j
j
i
i
! Org. -N -
/ NH4-*--N
/ N03~ -N
/ NO 2" -N
/
'~. J- T ^.i
313 6
300
200
O
100
Incubotion Time (months)
Figure 32. Soluble nitrogen in displaced soil solutions from sludge amended and con-
trol Ottokee sand incubated at field capacity (Experiment IV, summer-
autumn).
-------
00
3000
E
"S.2000
a.
^^
c
0)
o>
2
z
1000
224 Metric Ton
-
' ป
/
/
/
/
/'
/
/
j'
t
i
*\ -'
/ \ /
/ \ ;
' \
i \
/ ;\
-/ ' \
/ / V
1 i \
/ !
'
/ N
X
/ X
ซ...
r / "-...! |
90 Metric Ton
. "~' *
^ " """
/
/
/
/'
A /
/ \
/ ;\
/ / \
/ / x
' x
Control
'
Org. -N
NH4+ -N
N03~ -N
N02~ -N :
-
-.**
.
"'""
/.
/
/'
/"
/'
/
/'
?><* i _4
300
. g-
X
200 S
^
c
0)
o*
o
k.
^
z.
100
1
.
0 I
601 3 601
Incubation Time (months)
Figure 33* Soluble nitrogen in displaced soil solutions from sludge amended and con-
trol Ottokee sand incubated under saturated conditions (Experiment IV,
summer-autumn).
-------
vo
3000
^2000
o>
o
1000
224 Metric Ton
_. -o
o
90 Metric Ton
Control
, Org. -N
/ NH4+ -N
N03~ -N
NOsf -N
300
6
o>
200 ~
o
100
601 3 601
Incubotion Time (months)
Figure 34. Soluble nitrogen in displaced soil solutions from sludge amended and control
Celina silt loam incubated at field capacity ( Experiment I, autumn-winter.
-------
00
o
3000
o>
^2000
o>
o>
o
1000
224 Metric Ton
T-. 90 Metric Ton
\
"*"
i
i
H
Control
Org. -N
NH4+-N
N03~ -N
- -N
400
350
'275
E
V.
O>
2OO Jj^
c
o
100
601 3 601
Incubation Time (months)
Figure 35. Soluble nitrogen in displaced soil solutions from sludge amended and
control Celina silt loam incubated under saturated conditions (Experi-
ment I, autumn-winter).
-------
00
3000
E
?200G
IOOC
224 Metric Ton
90 Metric Ton
, -------
Control
Org. -(VI-
NO 3- -N
N02~ -N-
300
E
Nป
200
c
O)
o<
o
100
601 3 601
Incubation Time (months)
Figure 36. Soluble nitrogen in displaced soil solutions from sludge amended and. control
Celina silt loam incubated at field capaicty ( Experiment II, winter-spring).
-------
3000
E
o<
2000
c
0)
o>
o
00
1000
224 Metric Ton
- 300
- 200 -
o>
0 I
601 3 601
Incubation Time (months)
a>
o>
o
Figure 37. Soluble nitrogen in displaced soil solutions from sludge amended and control
Celina silt loam incubated under saturated conditions (Experiment II, winter-
spring) .
-------
oo
u>
4000,
3000
-~
"E
X
o>
32000
c
0>
o
2
1000
_,
224 Metric Ton
O
r ' ^
/
/
/
/
/
/
/
/
j
f
i
i
i
i
i
i
t
1
9- '
/ / -^
-. / j ^
t V.
.-.'.ป '" -i- y^ , J i
90 Metric Ton
*
x-
/<
/
/
/
/
/
/
j'
f
j
j
1
i
!
/
/
-^..^.ir-.^-^,,^ i
Control
.. -
." *
/'
/
/
'
/
S
/
/
i
i
i
i
i
! Org. -N -
f N03~ -N
N02~ -N-
,500
300
~~.
e"
o>
2003
c
o>
o
w.
Z.
100
0 I
601 3 601
Incubation Time (months)
Figure 38. Soluble nitrogen in displaced soil solutions from sludge amended and control
Celina silt loam incubated at field capacity (Experiment III, spring-summer).
-------
00
3000
^>
e
?2000
c
0)
o>
o
Z
1000
(
224 Metric Ton .
'
x
/
/
f
/
i
i
i
i
i
i
i
) 1 3 6(
90 Metric Ton
*
/"\
/ \
1 \
\
\
! \
! \
! \
! \
'-y*X / \
$^=^
313 6C
Control
dm W
NH4+ -N
N03~ -N
N02~ -N
-
r~ \
\
\
\ ^
\
\
\
\
. \
) 1 3 6
i
300
i
'
2;
E ;
N. !
200 ~ ;
c
01
a*
o ;
"z.
100
'
i
i
i
1
!
i
Incubation Time (months)
Figure 39. Soluble nitrogen in displaced soil solutions from sludge amended and control
Celina silt loam incubated under saturated conditions (Experiment III, spring-
summer) .
-------
oo
Ul
4OOOi
3500
E
3000
*""
I
^2000
c
-------
oo
3000
E
X
o>
-2000
c
V
o>
0
.t:
2
1000
224 Metric Ton
.**
,^
- f ^.-'''
i
i
i
i
.'
_.
i
i
i
i
i
K.
i >-| . ,
90 Metric Ton
f.
: \
j \
\
' \
.' \
.' \
' \
/ \
A .' \
/ \ / \
/., V \
/ V\ *
/ / \\
L ; X
^V' V ,
Control
urg. N "
NH4* -N
N03" -N
N02" -N
p
i\
i \
f \
\
\
\
\
\
\
\
\
^L \
..Vrs^^-i.^^
300
^^
I
^
200 jฃ
c
0)
o>
o
z
100
0 I
60 I 3 60 I 3
Incubation Time (months)
Figure 41. Soluble nitrogen in displaced soil solutions from sludge amended and control
Celina silt loam incubated under saturated conditions (Experiment IV, summer-
autumn) .
-------
00
150
100
50
"~*
E
o> 0
~ 250
1,225 =
2 150
z
100
50
0
" 224 Metric Ton
-
ฎx
/ X
/ X
/' 'x
! ^- -*
,
/
/'
x-x
X*
/ป'
x'
.x'
D 1 3 6
" 90 Metric Ton
. . .
-
^t
Ci^r--^
r *
^y
/x
/
/
/
j
/
013 6
ncubotion Time ( months
Control i
N03-
NH4+ -o
"5
3
_ 0
, ., .ฃ -. a^
-
' >, '
"o
o
a.
.* 3
'' 5
x' .ป
S li
013 6
)
Figure 42. Soluble ammonium and nitrate nitrogen in displaced soil solutions from sludge
amended and control Paulding clay (Experiment I, autumn-winter).
-------
00
oo
150
100
- 50
V.
O>
a.
~ 0;
c
a, 200
o 150
.ฑ
z
100
50
0
0
. 224 Metric Ton
-
x' '--..^
_x' '""
* **
/
-
/
/
1 3 6C
_ 90 Metric Ton
x'
x'
xฐ
x'
X
.'
//
. I'll , ^
~ /\
/ \
/ \
/ 'N
1 \
\
\
\
/ \
/ \
V _ '^
13 6C
_ Control
. ^3
O
3
O
-
>\
o
o
Q.
O
O
T>
Q)
U.
- -'*
> 1 3 6
Incubotion Time (months)
Figure 43. Soluble ammonium and nitrate nitrogen in displaced soil solutions from sludge
amended and control Paulding clay (Experiment II, winter-spring).
-------
The pattern of nitrogen accumulation in the Celina silt loam differed
from the Ottokee sand in several ways. First, the concentration of
ammonium nitrogen never approached that of the Ottokee sand, although
values as high as 500 ug/ml were found early in the incubation period.
Lower levels of NH4"1" in the soil solution would be expected because of
the higher cation exchange capacity of the Celina soil. Second, soluble
organic nitrogen normally reached insignificant levels after 1 months
incubation. Third, nitrite was found in a few of the soil columns above
the levels which might be considered normal. Fourthj nitrification was
more extensive in both the sludge amended and control dolls at field
capacity. However, there was still evidence of a partial inhibition of
nitrification of about a 1 month's duration. Lastly, the lower concen-
tration of nitrate in the saturated soils, as well as the decreases in
nitrate with time of incubation provide presumptive evidence for active
biological denitrification.
Quantities of ammonium and nitrate nitrogen in the displaced soil solution
from the Paulding clay are plotted in Figures 42 and 43. Data is not given for
Experiments III and IV because these studies were terminated before the
entire 6 month incubation period was complete. Nitrate nitrogen was found
to be the only significant form of soluble nitrogen in the Paulding clay.
Ammonium nitrogen was present at a concentration of less than 10 ug/ml.
Low concentration of soluble NH4+ would be expected in soils with a high
exchange capacity such as the Paulding. It is also apparent that the
concentration of soluble nitrogen found is considerably lower than in
comparable treatments for the Celina and Ottokee soils. In general and
as expected, the quantity of nitrate found when the soil was incubated at
field capacity was greater than under saturated conditions. This could
reflect either decreased nitrification or increased denitrification under
conditions of reduced aeration. As shown previously for the other two
soils, nitrification was again inhibited for about a 1 month period.
An attempt was made to estimate the percentage of the sludge organic nitrogen
which was found in soluble form after 6 months incubation. The calculated
values shown in Table 25 are corrected for soluble nitrogen in the control
soil and for ammonium nitrogen originally present in the sludge. Data were
not given for the Celina and Paulding soils incubated under saturated
conditions since the quantity of soluble nitrogen under these conditions of
reduced aeration would not be representative. Mineralized nitrogen appearing
in the soil solution ranged from 0.0 to 32.0% of the organic nitrogen
present in the sludge. The apparent lack of mineralization in the Paulding
clay must be interpreted by considering that nitrate accumulation will be
the net result of the opposing processes of nitrification and denitrification.
Net accumulation could be expected to be low in a fine textured poorly
aerated soil such as the Paulding clay. In addition, these data do not
allow us to determine the actual mineralization of sludge organic nitrogen,
since appreciable quantities of NH4+ nitrogen may be on the soil exchange
sites. The lower value in the Ottokee sand is a result of reduced microbial
activity because of limiting moisture. The higher value in the Celina silt
loam probably represents a maximum value associated with optimal conditions
for nitrification with little if any denitrification. Whether or not this
value represents the actual mineralization of nitrogen would depend on
89
-------
Table 25. PERCENTAGE OF THE ORGANIC NITROGEN OF ANAEROBICALLY DIGESTED
SEWAGE SLUDGE APPEARING IN THE SOIL SOLUTION AFTER 6 MONTHS INCUBATION.
Soil & Treatment
Sludge amendments (Metric Ton/ha)
224 90
Ottokee Sand- field capacity
(Data from Figure 32)
Ottokee Sand-Sat.
(Data from Figure33)
3.3%
10.0%
11.8%
13.4%
Celina silt loam-field capacity
(Data from Figure 40)
16.4%
32.0%
Paulding clay- field capacity
(Data from Figure 42)
0.0%
0.0%
90
-------
the amount of NH4 remaining on the exchange sites. Since no attempt
was made to measure exchangeable ammonium we cannot answer this question.
Spectrographic Analyses
The last group of analyses carried out on the displaced soil solution
were those for various nutrient cations and anions. Although spectro-
graphic analysis was routinely made for 15 elements (P, K, Ca, Mg, Na,
Si, Mn, Fe, B, Cu, Zn, Al, Sr, Ba, and Mo) only 6 elements (Ca, Mg, Na,
Cu, Mn, and Zn) showed differences in concentration which were attribu-
table to the various soil treatments. Three of these, Cu, Mn, and Zn
are normally present in soil solutions below the limit of detection by
spectrographic analysis. The addition of sewage sludge to the soil and
concomitant incubation and sludge degradation increased the concentra-
tion of these elements sufficiently to exceed the limits of detection.
Solution phosphorus was not increased, even though rather large quanti-
ties of phosphorus were added with the sludge amendments. This fact
illustrates the effectiveness of adsorption and precpitation reactions
in reducing the concentration of soluble phosphorus in soils.
Data showing the concentration of Ca, Mg, and Na in the displaced soil
solution are given in Tables 26, 27, and 28. It is apparent that the
addition of sewage sludge increases the concentration of all three
elements appreciably. The concentration of all three elements, but
particularly Ca also increased with incubation time, which may reflect
altered solubility and mineralization associated directly or indirectly
with microbial activity eg. increase in H+ associated with nitrification.
The soil solution from the Paulding clay was considerably more dilute
than the solutions from the Celina or Ottokee soils which were about equal.
We can expect that the large amounts of Na, Mg and Ca contained in sewage
sludge will participate in normal exchange reactions in soil. Once steady
state equilibrium is reached rather large concentrations of these elements
may be carried downward in the soil profile into groundwater supplies.
The concentrations of Zn, Cu and Mn in the displaced soil solutions are
shown in Tables 29, 30, and 31. As mentioned previously in this section,
these elements are seldom present in the soil solutions at concentrations
detectable by an emission spectrograph without prior concentration. The
presence of detectable concentrations of Zn, Cu and Mn in the soil solution
from sludge amended soils reflects the influence of sewage sludge in
supplying these elements to the soil. The concentration in solution at
any one time will reflect various microbial reactions such as oxidation
reduction, mineralization, production of soluble chelates, or indirectly
by changing the soil pH.
Manganese solubility is increased by conditions which would enhance the
reduction of MIT"4" to the more soluble Mn^+ . In this study, reducing
conditions associated with saturated moisture conditions and increased
levels of organic substrate would seem to explain the increased presence
of Mn in the soil solutions (Table 31). Soluble Cu (Table 30) was highest
in the Ottokee sand which also had the highest concentration of soluble
organic matter (Table 24). Increased solubility of Zn may reflect both
the accumulation of soluble organic matter as well as a reduction of soil
pH associated with active nitrification particularly in the Celina silt
91
-------
Table 26. CONCENTRATION OF Ca IN DISPLACED SOIL SOLUTIONS FROM SLUDGE AMENDED SOILS.
VO
NJ
Concentration of Ca (mg/ml)
Incubation Field Capacity
Soil
Ottokee
S'and
Celine
Silt
Loam
Paulding
Clay
Time
(Mo.)
0
1
3
6
0
1
3
6
0
I
3
6
Control
200
318
476
1111
940
1034
1280
2052
200
651
728
218
90 Ton
2413
1745
3061
5156
1700
6346
8083
7240
200
583
1370
250
224 Ton
2370
1010
1311
1965
3645
3715
6641
6695
1015
826
1298
1648
Saturated
Control
200
213
254
445
1210
1198
755
. 589
280
621
557
330
90 Ton
725
1940
5561
7146
1975
3862
5505
3130
355
508
930
1330
224 Ton
1465
1260
2773
4485
2140
4632
5665
8620
1965
1169
1918
2693
a Values given are the means for displaced soil solutions from duplicate columns for Experiments
I-IV. .
-------
Table 27. -CONCENTBATION OF Mg IN DISPLACED SOIL SOLUTIONS FR@M
-------
Table 28. CONCENTRATION OF Na IN DISPLACED SOIL SOLUTIONS FROM SLUDGE AMENDED SOILS.
Q
Concentration of Na (mg/1)
Incubation
Soil
Ottokee
Sand
Celina
Silt
Loam
Paulding
Clay
Time
(mo)
0
I
3
6
0
1
3
6
0
1
3
6
Field Capacity
Control
70
< 20
< 20
-c20
30
24
23
35
53
49
52
48
90 Ton
170
201
220
235
125
150
134
178
50
44
82
50
224 Ton
195
208
223
241
170
201
209
244
80
49
100
90
Control
55
^20
< 20
< 20
< 20
34
35
24 '
65
38
77
48
Saturated
90 Ton
135
164
175
185
110
125
145
140
55
50
82
105
224 Ton
165
210
241
246
185
219
185
245
85
65
103
100
a Values given are the means for displaced soil solutions from duplicate columns for experiments
I-IV.
-------
Table 29. CONCENTRATION OF Zn IN DISPLACED SOIL SOLUTIONS FROM SLUDGE
AMENDED SOILS.
Incubation
Soil
Ottokee
Sand
Celina
Silt
Loam
Paulding
Clay
Time
(Mo.)
0
1
3
6
0
1
3
6
0
1
3
6
Concentration of Zn (mg/1)
Field Capacity
90 Ton
ND
2
3
7
ND
3
9
9
ND
ND
2
ND
224 Ton
ND
3
4
1
ND
3
11
11
ND
1
1
5
Saturated
90 Ton
ND
2
10
13
ND
1
3
1
ND
1
1
5
224 Ton
ND
5
5
10
ND
9
11
19
ND
3
1
5
ND= Concentration below detection limit of the emission spectrograph
a Values given are the means for displaced soil solutions from duplicate
columuns for experiments I-IV.
95
-------
Table 30. CONCENTRATION OF Cu IN DISPLACED SOIL SOLUTIONS FROM SLUDGE
AMENDED SOILS.
Soil
Ottokee
Sand
Celina
Silt
Loam
Paulding
Clay
Incubation
Time
(Mo.)
0
1
3
6
0
1
3
6
0
1
3
6
90
1
2
2
3
ND
1
1
1
ND
ND
1
ND
*a
Concentration of Cu ( mg/1)
Field Capacity
Ton 224 Ton
2
7
10
12
ND
2
2
2
ND
ND
6
ND
90 Tori
ND
1
1
1
ND
1
1
ND
ND
ND
ND
1
Saturated
224 Tori
ND
2
4
4
ND
1
ND
ND
ND
1
ND
2
ND = Concentration below detection limits of the emission spectrograph
a Values given are the means for displaced soil solutions from duplicate
columns for experiments I-IV.
96
-------
Table 31. CONCENTRATION OF Mn IN DISPLACED SOIL SOLUTIONS FROM SLUDGE
AMENDED SOILS.
Incubation Concentration of Mn (mg/1^
Soil
Ottokee
Sand
Celina
Silt
Loam
Paulding
Clay
Time
(Mo)
0
1
3
6
0
1
3
6
0
1
3
6
Field
90 Ton
5
6
11
21
6
5
4
7
3
4
7
3
Capacity
224 Ton
3
3
4
3
9
20
28
49
5
5
7
7
90
3
7
47
68
5
53
42
25
3
4
7
7
Saturated
Ton 224 Ton
4
6
12
31
5
40
56
76
17
8
.12
17
Values given are the means for displaced soil solutions from duplicate
columns for experiments I-IV.
97
-------
loam. The significance of increased solubility of Zn, Mn, and Cu on the
uptake of these elements by Kentucky 31 Fescue will be discussed in the
next section of this report.
PHASE 4i EFFECTS OF ANAEROBICALLY DIGESTED SEWAGE SLUDGE ON KENTUCKY
31 FESCUE
One highly significant factor affecting the utilization of soil for disposal
of sewage sludges is the effect of the sludge oil the growth of higher
plants. Potential problems might arise because of phytotoxic effects
associated with sludge organic matter itself, organic degradation products,
changes in the soil microflora, soluble salts, excess ammonium or nitrite
nitrogen, ion imbalances in soil, or toxic concentrations of heavy metals.
Phytotoxic effects might be expressed by one or more plant responses such
as reduced germination, sub-optimal plant growth, or changed chemical
composition. The studies reported in this section evaluated the influence
of anaerobically digested sewage sludge on the germination, growth, and
chemical composition of Kentucky 31 Fescue. Evaluations were made on
sewage sludge amended soils after 1, 3 and 6 month incubation periods at
differing soil moisture contents and temperatures. Data summarizing the
effects of anaerobically digested sewage sludge on germination of Kentucky
31 Fescue are shown in Table 32. Values for percentage germination for
individual monthly experiments were often highly variable with no consistent
trends associated with either the length of incubation or temperatures
during incubation. Mean values for germination for each 6 month experiment,
as well as the grand means, did show some differences, however. In general,
germination of fescue in sludge amended soils incubated under saturated
moisture conditions was as good or better than unamended control soils.
The only pronounced negative effect noted in saturated soils was in the
Ottokee sand at the 224 metric ton loading rate, Reduced germination under
these conditions was probably related to the high osmotic pressure of the
soil solution (See Table 20) or to the influence of sludge colloids in
altering the imbibition of H20 by the germinating seeds. The improvement
in germination for the 90 metric ton sludge amendments in the Celina and
Paulding soils under saturated conditions was probably related to the
amelioration of adverse conditions associated with excess H20 by soluble
salts or sludge colloids. '
The same two factors noted above, alone or in combination, are probably
responsible for the reduced germination of Kentucky 31 Fescue in sludge
amended soils kept at field capacity. Severe reductions in germination in
the Paulding soil cannot be associated with the osmotic pressure of the
soil solution (See Table 22) and may be related to the energy by which 1^0
is held by sludge colloids.
Excellent germination of Kentucky 31 Fescue in sludge amended soils under
saturated conditions certainly suggests that anaerobically digested sewage
sludge does not directly affect germination. Sludge induced secondary effects
on water availability to germinating seeds because of soluble salts or
sludge colloids may be significant, however.
98
-------
Table 32, EFFECT OF ANAEROBICALLY DIGESTED SEWAGE SLUDGE ON GERMINATION OF KENTUCKY 31 FESCUE IN SLUDGE AMENDED
SOILS. DATA EXPRESSED AS % OF CONTROL
VO
VO
Exp. Incub.
No. Time
(mo.)
I 1
3
6
Mean
II 1
3
6
Mean
III 1
3
6
."Hean
IV 1
3
6
Mean
Grand
Mean
0 day
Ottokee Sand
Sludge (metric ton/ha)
90T-FC
86
108
68
87
82
68
64
71
59
54
116
76
96
114
16
75
77
89
90T-Sat.
91
100
98
96
92
102
102
99
94
85
100
93
103
113
92
103
98
95
224T-FC
46
72
76
65
76
50
63
63
58
94
107
86
92
72
32.
65
70
64
224T-Sat.
30
94
92
52
81
82
93
85
87
94
.88
90
93
95
84
91
80
65
Celina
silt loam
Sludge (metric ton/ha)
90T-FC
106
101
103
103
95
91
110
99
101
97
85
94
93
94
11
-68
91
88
90T-Sat
89
110
112
104
94
98
94
95
83
97
122
101
97
98
146
112
103
84
. 224T-FC
87
97
104
99
69
71
88
76
89
70
81
80
100
69
14
61
79
90
224T-Sat.
105
99
97
100
74
95
104
91
86
110
89
95
87
94
132
104
98 t .
65
Eaulding
Clav
Sludge (metric ton/ha
90T-FC
82
33
65
60
79
76
74
76
86
81
_ _
84
94
-'
74
62
90T-Sat.
107
96
124
109
93
no
102
102
145
96
~
121
135
-
112
106
224T-FC
71
37
46
51
62
45
52
53
66
36
L.
51
67
. -
'
53
27
224T-Sat.
103
50
104
86
113
88
89
97
146
63
I
105
130
-
98
98
-------
Data on the effect of anaerobically digested sludge on the dry matter yield
of 6 week old Kentucky 31 Fescue are shown in Tables 33 and 34. It is of
particular significance that sludge amended soils usually gave higher dry
matter yields of fescue than control soils (Ratios>10 in Table 33). The
exceptions can in most cases be related directly to poor seed germination
for reasons discussed previously (See germination data, Table 32). Likewise,
there were many instances where positive yield responces occurred in sludge
treatments even with reduced germination.
Fescue growing in sludge amended saturated soils produced considerably more
dry matter than those maintained in soils at field capacity. Visual
observations of fescue growing in sludge amended soils at field capacity
confirmed that conditions were less than desirable for maximum growth.
Typical plants were bluish-green, stunted, with necrotic leaf tips. In
contrast fescue growing in saturated soils was dark green, vigorous, with
no apparent necrosis or chlorosis. It should be emphasized, however, that
saturation of the soils in the growth chamber did not result in as poor
root aeration as would be true under natural soil conditions. Poor plant
growth in the sludge amended Ottokee and Celina soils at field capacity
could very well be associated with salt accumulation to toxic levels.
Specific conductance greater than 4 mmho/cm are generally considered to
restrict plant yields. Measured specific conductance values ranging between
11.1 and 21.8 mmhos/cm were obtained for displaced soil solutions from
these two soils (Tables 20 and 21). However, salt accumulation to toxic
levels can not be the total reason since specific conductance greater than
10 mmho/cm was rather commonplace in even the saturated soils in 224 ton
sludge amended soils without noticeable effects on plant growth. Further-
more, the soil solutions displaced from the Paulding clay had specific
conductances below 4 mmho/cm but plant growth was still affected adversely
in soil maintained at field capacity.
The fertilizer value of anaerobically digested sewage sludge is readily
apparent if one looks at the yield response to sludge amendments in the
soils maintained at saturated moisture conditions. However, it also appears
that near maximum yields were attainable at the lower 90 metric ton/hs
amendment with little yield increase attributable to the 224 metric ton/ha
amendment.
Plant analysis provided information on ion uptake by Kentucky 31 Fescue
from sludge amended soils after incubation for 1, 3 and 6 months. Nitrogen
and sulfur data are summarized in Table 35. These data are incomplete
because growth of fescue was so poor in many of the treatments that insuffi-
cient plant material was available for one or more of the analyses. Not
surprisingly plant nitrogen and sulfur were directly related to the quantity
of sludge added. Plant nitrogen was also lower in the saturated soils than
in soils incubated at field capacity. This may in part reflect the dilution
effect within the plant itself because of the better growth in saturated
soils compared to those kept at field capacity. Perhaps more important, however,
is the reduced availability of soluble nitrogen in saturated soils because
of an increase in biological denitrification, and reduced nitrification.
Phosphorus was also higher in Kentucky 31 Fescue grown in sludge amended
soils than in control soils (Figure 44). The one exception was plants grown
100
-------
Table 33. EFFECT OF ANAEROBICALLY DIGESTED -SFWAGE SLUDGE ON 0RY MATTER YIELD OF KENTUCKY 31 FESCUE, DATA EXPRESSED
AS RATIO SLUDGE TRT.
CONTROL
Exp. Incub.
No. Time
(Mo.)
I 1
3
6
Mean
II 1
3
6
Mean
III 1
3
6
Mean
IV 1
3
ฃ-
Mean
Grand
Mean
0 Day
Ottokee Sand
Sludge (metric ton/ha)
90T-FC
4.3
4.5
6.5
5.1
6.7
1.6
2.7
3.7
2.2
1.8
3.7
2.6
2.0
3.7
2.0
2.6
-4.5
2.5
90T-Sat.
16.1
11.9
5.4
11.1
11.7
8.8
9,0
9.8
7.3
6.9
6.8
7.0
6.7
7.6
3.6
6.0
,8.5
9.8
224T-FC
2.9
3.0
5.8
3.9
7.4
1.4
1.6
3.5
3.2
2.6
5.3
3.7
1.6
4.4
0.7
2.2
3.3
0.8
224T-Sat.
13.7
10.1
5.2
9.7
14.7
6.3
9.6
10.2
16.2
9.7
7.6
11.2
4.5
9.0
3.8
5.8
9.2
5.7
Celina Silt Loam
Sludge (metric ton/ha)
90T-FC
3.2
2.3
3.7
3.1
3.6
2.6
3.0
3.1
3.3
2.9
3.6
3.2
2.7
1.5
1.2
1.8
2.8
2.0
90T-Sat.
4.9
8.3
5.7
6.3
'3.6
5.6
6.3
5.2
3.9
5.7
2.9
4.2
3.3
5.6
1.9
3.6
4.8
7.3
224T-FC
2.3
3.3
2.5
2.7
3.7
3.1
2.8
3.2
3.5
2.1
3.6
3.1
2.4
1.8
0.2
1.5
2.6
2.8
224T-Sat.
17.6
6.5
15.0
13.0
6.0
11.5
15.8
11.1
8.3
13.8
13.9
12.0
7.5
13.3
10.3
10.4
11.6
9.8
Paulding
Clay
Sludge (metric ton/ha)
90T-FC
2.0
3.5
2.7
2.7
2.5
2.1
2.6
2.4
2.1
2.3
2.2
1.6
2.4
3.5
90T-Sat.
2.7
1.0
2.6
2.1
2.9
2.8
2.3
2.7
2.7
0.7
1.7
6.5
2.7
3.2
224T-FC
1.7
1.0
1.0
1.2
1.2
0.9
0.9
1.0
0.9
0.4
0.7
0.7
1.0
1.7
224T-Sat.
3.4
0.8
2.1
2.1
3.5
2.9
2.9
3.1
3.1
0.9
2.0
7.3
3.0 -
3.7
-------
Table 34. EFFECT OF ANAEROBICALLY DIGESTED SEWAGE AND SOIL MOISTURE
STATUS ON YIELD OF KENTUCKY 31 FESCUE. DATA EXPRESSED AS THE MEAN
OF DRY WEIGHT (g) FOR THE L, 3, & 6 MONTHS INCUBATION
Soil
Ottokee
Sand
Celina
Silt
Loam
Paulding
Clay
Exp.
No.
I
II
III
IV
Mean
I
II
III
IV
Mean
I
II
III
IV
Mean
Field Capacity
0
0.049
0.068
0.069
0.067
0.063
0.154
0.128
0.225
0.216
0.181
0.200
0.237
0.2713
0.055a
0.191
90T
0.237
0.217
0.171
0.193
0.205
0.462
0.383
0.746
0.421
0.503
0.498
0.578
0.588a
0.089a
0.438
224T
0.174
0.187
0.251
0.182
0.199
0.432
0.391
0.709
0.382
0.479
0 . 2 68
0.242
0.224s
0.040a
0.194
0
0.125
0.116
0.207
0.246
0.174
0.091
0.087
0.216
0.224
0.154
0.895
0.776
1.026a
0.3573
0.764
Saturated
90T
1.206
1.127
1.439
1.417
1.297
0.628
0.468
0.820
0.799
0.679
1.656
2.003
1.381s
2.333a
1.843
224T
1.065
1.152
2.152
1.439
P
1.452 U
1.003
1.027
2.491
2.212
1.683
1.86&
2.358
1.554's
2.613a
2.097
Data for Paulding Clay is incomplete since no data was available
for Exp. Ill, 6 mo. and Exp IV, 3 and 6 mo.
102
-------
Table 35. NITROGEN AND SULFUR CONTENT OF KENTUCKY 31 FESCUE GROWN IN
SLUDGE AMENDED SOILS.3
Soil
Treatment
Control-FC
90 Ton-FC
224 Ton-FC
Control-Sat.
90 Ton-Sat.
224 Ton-Sat.
Ottokee
N%
-,
2.70
3.20
1.30
1.69
2.51
Sand
S%
.
0.34
0.57
0.10
0.41
0.52
Celina
N%
1.91
3.39
3.41
1.17
1.39
2.46
Silt Loam
S7o
0.29
0.34
0.28
0.64
0.46
Paulding
N% .
1.57
3.24
3.52
1.00
2,03
2.67
Clay
S%
.
-
.
0.29
0.34
Data shown are the means for a particular soil sludge-soil moisture
treatment over all incubation periods and experiments.
bFC = Field capacity
cSat. = Saturated .
103
-------
1.0
0.8
0.6
0.4
0.2
' 0
1.0
0.8
ฃ0.6
ฐ-0.4
0.2
0
1.0
0.8
0.6
04
0.2
O
Field Copocity .
Ottok
-
_\
f^^^^^^l
-
Saturated
ee Sand
- ^
i i i
' 224 Metric Ton
90 Metric Ton -- Celine Silt Loom
Control P
-
-X
i i i
-
-'*'^
V "*"* "
Poulding Cloy
-
i i i
-
3 601 3
Incubation Time (months)
Figure 44. Phosphorus content of Kentucky 31 Fescue grown
in sludge amended soils.
104
-------
in the Ottokee sand at field capacity. Hei*.the obviously poor growth
nay have altered the normal plant physiological processes and affected
P uptake.
Potassium uptake by Kentucky 31 Fescue was unaffected by sludge additions
to soils (Figure 45) except for a small positive response in the Ottokee
sand. Since anaerobically digested sewage sludge adds relatively little
K to soils this overall low response was expected. The Ottokee sand was
so low in potassium (note the low K concentration in the control plants)
that the addition of even low concentrations in the sludge resulted in
increased plant uptake of K.
Plant content of Mg and Ca were largely unaffected by sewage sludge
amendments (Figure 46 and 47). This was true even though the concentration
of Ca and Mg in the displaced soil solutions (Tables 26 and 27) was increased
considerably by sludge additions. Perhaps the increased uptake of Na by
the Fescue (Figure 48) was responsible for a decreased uptake of the divalent
Ca and *Mg ions.
Sodium uptake by Kentucky 31 Fescue was increased consistently in all soils
and at all moisture contents (Figure 48). These data reflect the relatively
large additions of Na with the sludge. The Na content of the displaced
soil solution of the Ottokee sand and Celina silt loam increased about
6-8 fold because of the sludge amendment (Table 28). The increase in the
soil solution of the Paulding clay was neglible but the activity of exchangeable
sodium undoubtedly remained high enough to provide large quantities of Na
for plant uptake.
The three nutrient elements Cu, Zn, and B showed similar uptake patterns
from sludge amended soils. Decreased uptake of all three elements was
associated with an increase in soil moisture saturation and reduced aeration
(See Figures 49, 50, and 51). The greatest reduction in uptake was evident
in the Paulding clay, while Celina silt loam gave an intermediate response.
In the coarse textured Ottokee sand, water saturation had no effect on the
uptake of Zn, Cu and B. Reductions in Zn, Cu and B in corn grown on fine
textured soils were previously observed by Lai and Taylor (1970) in lysimeters
with shallow water tables or those flooded intermittently. These authors
attributed this reduced uptake to decreased ion solubility caused by their
coprecipitation with soluble Al and Fe in soils under reducing conditions.
In this study other explanations seem more plausible. Since there is a
direct relationship between microbial activity as determined by C02 evolution
and the uptake of Zn, Cu and B, we can speculate that the response is due
to mineralization of these elements bound to sludge organics or by the
greater production of low molecular weight organic ligands which would
influence ion, solubility and availability. Alternatively, an indirect
effect of microbial activity, namely the reduction of pH by nitrification
and the increased solubility of Zn, Cu and possibly B, may also be of
significance. Unfortunately there does not seem to be a relationship
between the concentration of Zn and Cu in the displaced soil solution (Tables 29
and 30) and plant uptake of these elements.
105
-------
5.0
4.0
2.0
1.0
0
Field Capacity
Ottokee Sand
Saturated
nf\ ~'~ """"
\
i- \
5.0
4.0
3.0
2.0 fcr
1.0
0
224 Metric Ton
90 Metric Ton Celino Silt Loam
Control
^
. r.-s-**^ = -^ - *
Poulding Cloy
01
3 601 3
Incubation Time (months)
Figure 45. Potassium content of Kentucky 31 Fescue grown
in sludge amended soils.
106
-------
Field Copocity
Ottokee Sond
Saturated
2.0
1C
.6
1.2
0.8
0.4
0
Poulding Cloy
x'*
X
, X
X
J>"~~~^^^""^"ฐ
5*>- -^-. '
s ^>'
r
1 1 1
" 224 Metric Ton
90 Metric Ton
Control
-
^**-<^
^o.^^ '3
-------
Field Capacity
Ottokee Sand
Saturated
3 601 3
Incubation Time (months)
Figure 47. Magnesium content of Kentucky 31 Fescue grown
in sludge amended soils.
108
-------
0.3
0.2
Field Capacity Saturated
Ottokee Sand
"^s-rr
Cell no Silt Loam
0.2 v
O.I
0
0.3
0.2
O.I -'
" 224 Metric Ton
90 Metric Ton Poulding Cloy
Control ""
0
3 601 3
Incubation Time (months)
Figure 48. Sodium content of Kentucky 31 Fescue grown in
sludge amended soils.
109
-------
250
150
50
0
250
E
~ 150
c
M
50
0
250
150
50
O
Field Copocity
Ottok
*\ -*>~
\ ^""^^''^
x. * .''"
i i i
Celine
X--^
^ ^' ' '"-
t- ,
1 1 1
^-;.tpฐl
' 0. ** '
Saturated
ee Sand
_---.
-.'^'-^
i r i
ailt Loam
-
- .xX"~~ -
I-*"
^ -
i^_-ป .'
1 1 1
ilding Cloy
224 Metric Ton
90 Metric Ton
Control
_. -ป
^-^^3ฃ^ t
01 3 601 3
Incubation Time (months)
Figure 49= Zinc content of Kentucky 31 Fescue grown in
sludge amended soils.
110
-------
30
25
20
15
10
5
0
30
25
~ 20
E
ฃ 15
= 10
o
5
0
30
25
20
15
10
5
0
Field Copocity
Ottok
-x'
1 1 1
Saturated
ee Sand
-
Celine !
^^^
Silt Loam
^r _
n
Pould
r
-
ng Clay
224 Metric Ton
90 Metric Ton - -
Control
i i i
3 6013
Incubation Time (months)
Figure 50. Copper content of Kentucky 31 Fescue grown in
sludge amended soils.
Ill
-------
a.
o.
m
300
200
100
0
3OO
200
100
0
300
200
100
0
Field Capacity Saturated
Ottokee Sand
Celina Silt Loam
Paulding Clay
224 Metric Ton *
90 Metric Ton
Control
01 3 601 3
Incubation Time (months)
Figure 51. Boron content of Kentucky 31 Fescue grown in
sludge amended soils.
112
-------
Manganese availability and uptake by Kentucky 31 Fescue is shown in
Figure 52. Although there is a trend for the plant uptake of Mn to
follow that of Zn, Cu, and B discussed previously, there are enough
inconsistencies to make interpretation difficult. Also disconcerting
is a lack of correspondence between the concentration of Mn in the dis-
placed soil solution (Table 31) and Mn uptake. This may be attributable
to the ease of oxidation-reduction reactions of Mn in soil influenced
by microbial activity, organic matter content, and water saturation. Data
for plant analysis of Al, Fe, Sr, Ba, and Mb were also obtained but are
not included because there were no differences between plants grown in
sludge amended or control soils.
113
-------
250
150
50
0
250
I 150
a.
Field Capacity Saturated
Ottokee Sand
Celina Silt Loam
- V-'
250
150
50
0
224 Metric Ton
90 Metric Ton
Control
Paulding Clay
01 3 601 3
Incubation Time (months)
Figure 52. Manganese content of Kentucky 31 Fescue grown
In sludge amended soils.
114
-------
REFERENCES
1. Anonomyous. Waste Engineering. Oct. 1962. Survey of
design trends and developments for small sewage treat-
ment plants in past decade, pp 520-523.
2. American Public Health Association. 1971. Standard
Methods for the Examination of Water Sewage, and
Industrial Wastes. 13th ed., American Public Health
Association.
3. Bremmer, J.M. 1965. Inorganic Form of Nitrogen in
Methods of Soil Analysis. Part 2. Chemical and Micro-
biological Properties. C. A. Black (ed), American
Society of Agronomy, Monograph No. 9, American Society
of Agronomy, Inc., Madison, Wisconsin.
4. Burd, R.S. 1968. A study of sludge handling and disposal.
Water Pollution Control Research Series. Publication No.
WP-20-4 U.S. Dept. of Interior, R. W. PCA.
5. Carolan, R. 1948. Modification of Graham's method for
determining soil organic matter by colorimetric analysis.
Soil Sci. 66:241.
6. Cold Regions Research and Engineering Laboratory Staff.
1972ปWastewater management by disposal on the land. Special
Report 171, CRREL, Corp of Engineers, U.S. Army, Hanover, N.H.
7. Collins, C.H. and P.M. Lyne. 1970. Microbiological Methods.
3rd edition, University Park Press, Baltimore, Md.
8. Ewing, B.B. and R. I. Dick. 1970. Disposal of sludge on
land. In Water Quality Improvement .by Physical and Chem-
ical Processes. E. F. Gloyna and W. W. Eckenfield, Jr. (ed),
pp 394-408. University of Texas Press, Austin.
9ป Franklin, R.E. and E. 0. McLean, 1963. Effect of electrolyte
concentration on Donnan systems and the resulting uptake of
cations by plants. Soil Sci. Soc. Amer. Proc. 27:137-141.
10. Fung, D.Y.C. and R. D. Miller. 1970ป Rapid procedure for the
detection of acid and gas production by bacterial cultures.
Applied Microbiol. 20:527-528.
115
-------
11. Geldreich, E.E., H.F. Clark, C.B. Huff. L.C. Best 1965.
Fecal-colifonn-organism medium for the membrane1 filter
technique. Journ. American Water Works Association 57:
208-214.
12. Hinesly, T.O., O.C. Braids and J.E. Molina. 1971. Agri-
cultural benefits and environmental changes resulting
from the use of digested sewage sludge on field crops.
Report SW-30d.,, U.S. Environmental Protection Agency.
13. Hinesly, T.O. R. L. Jones, and E.L. Ziegler. 1972.
Effects on corn by applications of heated anaerobically
digested sludge. Compost Sci. 13:26-30.
14. Jackson, M.L. 1958. Soil Chemical Analysis. Prentice-Hall,
Inc., Englewood Cliffs, N.J.
15. Jensen, V. 1968. The plate count technique. In The Ecology
of Soil Bacteria. TRG Gray and D. Parkinson, (ed.). Liver-
pool University Press, Liverpool, U.K.
16. Lai, R. and G.S. Taylor. 1970. Drainage and nutrient effects
in a field lysimeter study: II. Mineral uptake by corn.
Soil Sci. Soc. Amer. Proc. 34:245-248.
17. Larsen, S. and A. E. Widdowson. 1968. Chemical Composition
of soil solution. J. Sci. Fd. Agric. 19:693-695.
18. Martin, J.Po 1950, Use of acid, rose bengal and streptomycin
in the plate method for estimating soil fungi. Soil Sci. !
69:215-233.
19. McCoy, J.H. 1971. Sewage pollution of natural waters. In
Microbial Aspects of Pollution, G. Sykes and F.A. Skinner, \
(ed). Academic Press, N.Y. j
i
200 McLean, E.O. and R., E. Franklin, Jr. 1964. Cationic activities !
in clay suspensions and equilibrium dialyzates. Soil Sci. i
97:260-267. . . j
I
21. Miller, R.H. 1973. Soil as a biological filter. Proceedings
of a symposium on Recycling Treated Municipal Waatewater
and Sludge Through Forest and Cropland. Penn State University,
August 21-24, 1972. (In Press).
22. Personnel, A.R.S., U.S.D.A., 1972. Incorporation of
sewage sludge in soil to maximize benefits and minimize
hazards to the environment, First Progress Report, June
30, 1972.
116
-------
23. Reitemeier, R. F. and L.A. Richards. 1944. Reliability
of the pressure-membrane method for extraction of soil
solution. Soil Sci. 57: 119-135.
24. Richards, L.A. ed. 1954. Saline and Alkali Soils. Agri-
culture Handbook No. 60, U.S.D.A.
25. Stotzky G. 1965. Microbial Respiration. Itl Methods of
Soil Analysis. Part 2. Chemical and Microbiological
Properties. C.A. Black (ed) American Society of Agronomy,
Monograph No. 9, American Society of Agronomy, Inc.,
Madison, Wisconsin.
26. Thomas,R. E. and T.W. Bendixen, 1969i Degradation of
wastewater organics in soil. Jour. Water Poll. Control
Fed. 41:808-813.
27. Umbreit, W.W., R. H. Burris, and J.F. Stauffer. 1964.
Manometri? Techniques. Burgess Publishing Co., Minneapolis,
Minn.
117
-------
Appendix A
Sludge - Soil Extract Agar
l.Og Glucose
0.5g K2HP04
750 ml Sludge Extract*
100 ml Soil Extract**
150 ml Tap Water
Preparation of Sludge Extract - One liter of anaerobically digested
sewage sludge was autoclaved at 121ฐC for 30 minutes. The sus-
pension was filtered through Whatman #1 filter paper using
Celite filter aid.
JLJL
Preparation of jaoil Extract - One liter of tap water was added to
lOOOg of a fertile soil and autoclaved at 121ฐC for 20 minutes.
Colloids were flocculated with 0.5g CaC03 and the suspension
filtered through Whatman #1 filter paper.
118
------- |