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CONTRIBUTION OF MICROORGANISMS TO ZINC IMMOBILIZATION IN SOIL

BY

Bahram Zamani

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1983

ABSTRACT
CONTRIBUTION OF MICROORGANISMS TO ZINC IMMOBILIZATION IN SOIL

by

BAHRAM ZAMAN I

A soil perfusion system was used to determine Zn immobilization by
soil microorganisms in Rubicon sand (pH 5.9). A 17 mgkg'l (.26 mM) Zn
solution (320 mL) was perfused through 12.5 g of gamma-irradiated
(sterilized) or biologically active soil. Approximately 75% of the
perfusate Zn was inactivated by chemical and physical mechanisms. The
introduction of biologically active soil microorganisms and sterile
nutrient broth into a sterile soil perfusion system resulted in an
additional significant reduction in Zn concentration of the soil
perfusate. The level of Zn in the perfusate of the sterile perfusion
system remained constant (3.9 mgL’1 of Zn) during the same 72 hour
perfusion period where the Zn level in the perfusate of the biologically
active (ixunnilated) system decreased to 0.7 mgL“1 of Zn. The enhanced
immobilization represented over 90% of the Zn from the perfusion
solution indicating that microorganisms immobilized a fraction of Zn
(15%) in addition to that activated by chemical and physical mechanisms.

A soil sample was obtained from the soil perfusion column after
maximum fixation had been attained. This soil sample was diluted in
sterile water and surface plated on soil extract agar containing 652m.
After colonies of microorganisms developed, the agar plate was placed on
Kodak film X-OMAR-AR or NO-SCREEN (NS-2T) for autoradiography. The

colonies that accumulated sufficient levels of radioactive 652m to

expose the X-ray filnnwere identified by comparison with the developed
film. The colonies were isolated, grown in pure culture, and
reconfirmed as "zinc accumulators" by the autoradiographic plating
technique on 652h enriched agar.

MmN:of the isolated Zn immobilizing organisms were fungi,

identified as predominantly Penicillium species. Other Zn-immobilizing

 

fungi were Fusarium, Paecilomyces, Cladosporium, Cephalosporium, Mucor,

and Aspergillus spp. The most abundant Zn-immobilizing bacteria were

 

spore-forming Bacillus spp., and another unidentified gram—positive rod.

 

To Drs. Knezek and Dazzo

ii

ACKNOWLEDGMENTS

I sincerely thank Dr. B. D. Knezek and Dr. F. Dazzo with whom I
exchanged ideas throughout this research project. Their great interest,
valuable time and continual guidance made my educational and research
experience at Michigan state University very rewarding, has been greatly
appreciated and will always be remembered.

My special gratitude is extended to Drs. In S. Robertson, J. C.
Shickluna and F. Saettler for their valuable comments and to Dr. D.
Christenson for his help in the statistical design and analysis of data.

My sincere thanks are given to Drs. B. G. Ellis and L. W. Jacobs
for their participation and advice; to Dr. D. Penner and Mr. F.
Roggenbuck for their assistance in autoradiography and development of
X-ray films; to Dr. E. S. Beneke for his precious time and effort in the
identification of Zn-immobilizing fungi; to Drs. J. Funkhouser and R.
Nicholas for use of the variable flux gamma irradiator and for the
Special help of Mr. J. Carrick who facilitated the production of a
radio-isotope Zn (65Zn) at the Michigan State University nuclear reactor
laboratory.

The author is indebted to Mr. C. Bricker for his interest, concern
and assistance in all aspects of the research; Mr. J. Sherwood and Ms.
E. Hrabak are thanked for their help in the microbiology laboratory; and
appreciation is extended to Ms. B. Bricker for her thorough and critical
reading, reviewing and correction of the manuscript. Also, the aid and

secretarial skill of Ms. R. Burrough in the final typing and preparation

iii

of the dissertation is appreciated.
Appreciation is expressed to Dr. B. D. Knezek and Dr. D. D.
Harpstead for their financial support during a critical period of my

education at Michigan State University.

iv

TABLE OF CONTENTS
Page
LIST OF TABLES 0.09.0000....00.00.00.00.00.000000000000000...... v1

LIST OF FIGURES oooooooo00.000000000000000...00000000000000.0000 Vii

INTRODUCTION 0 O O O O O O O ..... O O O O O O O O O O O O O O ..... O O O O O O O 0 O 0 O O O O O O O O O 1
LITERATURE REVIEW 0 I O O O O O O O I O I O O O O O I O O O O O O O O O I O O O O O O O O O O O O O ..... 4
General Characteristics of Zinc ...................... ..... 4

Zinc Metalloenzymes and Effect of the Heavy Metals

on Enzyme Activity in Soil ......................... 6
Characteristics of Sewage Sludge Containing Zn and

Other Heavy Metals ................................. 9
Microbial Activity as Affected by Environmental

and Heavy Metal Contamination ...................... 12
Fixation and Availability of Heavy Metal by 3

Soil Component .......................... ..... ...... 17
Effects of Sterilization on Soil and

Microbial Response ................................. 21
Use of Soil Perfusion Apparatus in Studying Microbial

Processes in the Soil .............................. 25

MATERIALS ...................................................... 23
Soils ..................................... .......... ..... 28
Soil Perfusion System .........................Z... 28
Soil Perfusion Column and Perfusate ...................... 33
Energy and Nutrient Enrichment of Perfusate .............. 34
Agar Plate Preparation for Dilution and Counting ......... 34

METHODS ........................................................ 35
Soil Characteristics and Textural Analysis ............... 35
Inorganic Zn Analysis .................................... 35
Inhibitory (static) and Lethal (cidal) Effect of Zinc .... 37
Sterilization and Microbial Growth Inhibitors ............ 37
Uniformity of Perfusate Solution Mixing .................. 38
Soil Perfusion System Operation in Experimental Mode ..... 38
Statistical Procedures ................................... 39
Dilution and Counting of Soil Microorganisms ............. 40
Isolation of Zn Immobilizing Soil Microorganisms ......... 40
Isolation of Pure Culture of Microorganisms .............. 41
Identification of Zn Immobilizing Microorganisms ......... 42

RESULTS AND DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O O O O ........ I O O O O O O O 44
SUMMARY ..... O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O ....... O O O O O O O O O O O I 89
CONCLUSIONS 0 O I O O 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O O O 9]-

LITERATURE CITED ....OOOOO ....... 00......OOOOOOOOOOOOOOOOOIOOOI. 93

LIST OF TABLES
Page

Table l. Textural analysis of Rubicon sand and Brookston
Clay loam. OOOOOOOOCOCOCCOOOOOCOCOOOOOQOOCCOOOCOOOOOOOO 35

Table 2. Chemical characteristics of Rubicon sand,
Brookston clay loam and Houghton muck. ................ 36

Table 3. Filtration of the soil perfusate over time to

determine if Zn in the reservoir is present in a
particulate form. O...00.0.00...OOOOOOOOCOOOOOOOOO ..... 48

vi

LIST OF FIGURES
Page

Figure 1. Soil perfusion apparatus used in these studies.
Note the indicated column of packed soil. .......... 30

Figure 2. Three soil perfusion units connected in series.
Glass became tinted brown as a result of gamma
radiation. 0.00.00...00......OOOOOOOOOOOOOOOOOOOOOO. 32

Figure 3. The performance consistency of three different
soil perfusion units. A solution of Zn (5 mL
containing 5 000 mgkg"1 Zn) was added to each
soil column at time equal zero, and the Zn
concentration in the reservoir was determined at
intermittant intervals. ............................ 46

Figure 4. The influence of microorganisms and denatured
nutrient broth upon Zn immobilization in a soil
perfusion apparatus which was sterilized by
autoclaving. Percolate from the reservoir was
sampled periodically and analyzed for Zn by
atomic absorption spectrOphotometry. ............... 50

Figure 5. The influence of microorganisms and denatured
nutrient broth upon Zn immobilization in a soil
perfusion apparatus which was sterilized by
gamma radiation. Microorganisms were added to
one sterile treatment at the 68th hour with‘a
subsequent increase in Zn immobilization.
Percolate from the reservoir was sampled
periodically and analyzed for Zn by atomic
absorption spectrophotometry. ...................... 52

Figure 6. The influence of microorganisms and denatured
nutrient broth upon Zn immobilization in a
Rubicon sand soil column contained in a soil
perfusion apparatus which was sterilized by
gamma radiation. Percolate from the reservoir
was sampled periodically and analyzed for Zn by
atomic absorption spectrOphotometry. ............... 55

Figure 7. The influence of microorganisms and denatured
nutrient broth upon Zn immobilization in a
Brookston clay loam soil column contained in a
soil perfusion apparatus which was sterilized by
gamma radiation. PErcolate from the reservoir
was sampled periodically and analyzed for Zn by
atomic absorption spectrOphotometry. ............... 57

vii

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

10.

ll.

12.

13.

14.

15.

16.

17.

18.

The influence of microorganisms and denatured
nutrient broth upon Zn immobilization in a
Houghton muck soil column contained in a soil
perfusion apparatus which was sterilized by

gamma radiation.

Percolate from the reservoir

was sampled periodically and analyzed for Zn by

atomic absorption spectrOphotometry.

The influence of microorganisms and denatured
nutrient broth upon the Mn immobilization in a.
Rubicon sand soil column contained in a soil
perfusion apparatus which was sterilized by

gamma radiation.

Percolate from the reservoir

was sampled periodically for Mn by atomic

absorption spectrOphotometry.

Autoradiographic exposure of X-ray film by
isolated colonies of 65Zn-immobilizing soil

microorganisms.

Autoradiographic exposure of X-ray film by a

sterile control plate,

which represents the

background exposure of X-ray film by the 65Zn in

the culture medium.

Reconfirmation of 652n immobilization by a pure
culture of Penicillium using the autoradiography

 

plate technique.

Phase contrast
Zn-immobilizing

Phase contrast
Zn-immobilizing

Phase contrast
Zn-immobilizing

Phase contrast
Zn-immobilizing

Phase contrast
Zn-immobilizing

Phase contrast
Zn-immobilizing

Phase contrast
Zn-immobilizing

photomicrograph of a culture of
Penicillium £2. (x2652).

 

photomicrograph of a culture of
Paecilomyces s2, (x2652).

 

photomicrograph of a culture of
Fusarium_§p. (x2652). ..............
photomicrograph of a culture of

Cladosporium £2. (x2652).

 

photomicrograph of a culture of
Aspergillus §B° (x2652).

 

photomicrograph of a culture of
Mucor EB' (x2652).

photomicrograph of a culture of

RhiZOEus fl. (X2652) 0

viii

Page

59

62

66

68

7O

72

74

76

78

80

82

Figure 20.

Figure 21.

Page

Phase contrast photomicrograph of.a<uflture of
gram-positiye Bacillus sp. which immobilized Zn
(X6637). OOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOO0.0.0.0.... 86

Phase contrast photomicrograph of a culture of

unidentified, gram-positive rod which
1mm0bilized zn (X6637). o 00000. .00 oooooooo o ....... so 88

ix

INTRODUCTION

'Hm nfle of soil microorganisms in the inactivation of
micronutrients such as Zn by microbial immobilization in soil has been
considered to be insignificant when compared to chemical and physical
mechanisms of fixation. A significant microbial impact upon the
solubility of soil micronutrients that can readily undergo oxidation and
reduction, such as Fe or Mn, has been reported. However, the influence
on Fe and Mn solubility was probably due to chemical and physical
changes which were initiated by the change in the oxidation state of
micronutrients rather than by immobilization by soil microorganisms.
Likewise, the importance of soluble soil organic matter upon
Imicronutrient solubility in soil through complexation and chelation has
been reported by several investigators (Hodgson, 1963; Randhawa and
Broadbent, 1965; Stevenson and Ardakani, 1972; Stevenson, 1976, 1977 and
1982). According to the National Research Council (1979) newly formed
organic substances which are mobile (especially fulvic acids and
biochemical intermediates), can solubilize Zn and increase the metal's
availability to plants and other biological systems. A direct link with
microbial immobilization has not been established in these reactions and
the primary mechanisms are reported to be chemical and physical in
nature. Generally, data that define the magnitude of micronutrient
immobilization in soils are very limited, and data which describe the
relative importance of different microorganisms of the soil microbial

population upon micronutrient immobilization are extremely limited.

Zinc is a logical micronutrient choice for model investigations
involving microbial immobilization in soil because: it is an essential
micronutrient; it is not subject to oxidation and reduction under usual
soil conditions; it is relatively nontoxic (according to Brown et a1.,
1964, its contamination may cause illness with food poisoning); it has
been shown to be accumulated by and cause growth response in

Aspergillus; and there is considerable experience and background data on

 

soil fixation (KrauskOpf, 1956; Foy et a1., 1978; Reddy and Perkins,
1974). In addition, there is considerable current interest in the
environmental impact of Zn-contaminated waste materials such as
municipal sewage sludge and industrial wastes. Human health minimum
daily requirements and associated benefits of Zn supplementation of the
human diet as well as potential toxic influences on human and animal
systems have been the subject of many recent popular and scientific
literature sources. There is a growing recognition of the importance of
Zn in healjfliiand certain areas of potential research such as the
influence of Zn and other trace elements upon microbial activity in
digestive systems of livestock has not really been investigated.

A model approach to demonstrate the principles of the concept of
significant Zn immobilization by soil microorganisms was decided upon
and a system was needed that would include a soil in which usual Zn
chemical and physical fixation reactions were minimal and the
development of a population of soil microorganisms could be achieved
very rapidly under controlled environmental conditions. The combination
of Rubicon sand soil (which has a low cation exchange capacity, low

organic matter content and a pH of 5.9) with a soil perfusion system and

a Zn spiked perfusate provided an opportunity to test the hypothesis
that soil microorganisms can immobilize a fraction of Zn from soil
solution beyond that which would be fixed by chemical and physical
reactions on a short term basis.

Accumulated colonies which were isolated in "pure culture" on agar
slants were used as the source population to confirm that they were Zn
accumulators. Finally, preliminary attempts were made to identify the
genus of some major Zn accumulator colonies.

The Opportunity to identify, select and develop populations of
soil microorganisms that can selectively immobilize soluble Zn 111 soils
can have important practical benefits for environmental cleanup of Zn
contaminated soils. The processes of microbial immobilization could be
used to concentrate low levels of metals such as Cd by bacterial cells
(Kurek et a1., 1982), organic compounds as well as radioactive elements
from contaminated solution. Strandberg et al. (1981) used microbial
cells as biosorbents to accumulate uranium. The uranium was accumulated

extracellularly on the surfaces of Saccharomyces cerevisiae and

 

Pseudomonas aeruginosa and could be removed chemically. The cells could

 

then be reused as a biosorbent.

This study was undertaken to test the hypothesis that Zn could be
immobilized by soil microorganisms in quantities great enough to lower
the Zn levels in soil solution--especia11y when soils of low cation
exchange capacity (CEC) were amended with material high in Zn and
available energy and nutrient supply (carbon, nitrogen,1flmmphorus,

etc.)--such as when Zn enriched sewage sludge is added to sandy soils.

LITERATURE REVIEW

General Characteristics of Zinc

 

Zinc is a silvery metal that quickly tarnishes to a blue-gray
appearance. This color is due to an adherent coating of carbonate,
Zn2(OH)2CO3, which protects the underlying metal from further corrosion.
The metal is hard and brittle at ordinary temperatures but ductile and
malleable at loo-150°C (Nebergall et a1., 1972). Zinc has 18 electrons
in the underlying shells but only two in its outer shell therefore the
divalent oxidation state prevails. Coordination numbers of both 4 and 6
are quite common in the mineral structures and complexes of this element
despite its large size (0.74 A). Taylor (1964) stated the average
amount of Zn in the earth's crust was 70 mgkg"1 and in igneous rocks was
40 to 100 mgkg'l. Turekian and Wedepohl (1961) reported the Zn content
of sedimentary rocks (sandstone, limestone and shale) as being 16, 20
and 95 mgkg’l, respectively. Allaway (1968) reported the total Zn
content of the soils in the range of 10 to 300 mgkg'l, including the
majority of soils but certainly not the extremes (Zn content of soils
near sulfide deposits). Quartz is low in Zn, so sandy soils often
contain only 10-30 mgkg"1 total Zn (Lindsay, 1972a). Soil near highways
may be contaminated appreciably; the source of the Zn may be from the
wearing of tires containing ZnO and from emissions of motor oil to which
Zn-dithiOphosphite has been added; these sources of Zn are in addition
to industrial emanations (Lagerwerff and Specht, 1970). Klein (1972)
found that soil from an industrial area near Grand Rapids, Michigan, had

a mean content of 57 mgkg"1 Zn, whereas nearby agricultural and

residential areas had respective means of 22 and 21 mgkg‘1 Zn. The fact
that Zn occurs chiefly as the single sulfide mineral sphalerite (ZnS)
indicates that the metal is largely chalcophile in its behavior at the
earth surface. The transportation of Zn in solution is not a problem
except in solutions containing sulfide ion. Even in fairly alkaline
non-sulfide solutions, appreciable Zn can remain dissolved in the
presence of common anions, and the solubility is not affected by changes
in redox potential (Mortvedt, et a1., 1972). Hodgson (1963) stated that
a wide range of biochemical compounds and also organic substances like
humic and fulvic acids form stable combinations with Zn. The soluble
organic complexes have a profound effect on the mobility and
availability of Zn. Some of the Zn-organic complexes are so stable that
Zn is essentially unavailable to living systems, but these are rare.

One main group of organic compounds that form stable compounds
with Zn in the soil are direct biological products which originated in
living organisms and contain various organic acids, peptides, proteins
and polysaccharides. A second group includes organic polymers such as
humic and fulvic acid formed by secondary organic synthesis reactions
and result in more complex organic compounds (National Research Council,
1979). These two groups cannot always be clearly separated because Zn
can function as a linkage in binding these organic compounds and it can
be chelated by them (Stevenson and Ardakani, 1972). Lucas and Knezek
(1972) concluded that climatic and soil conditions were partly
responsible for Zn deficiency in crops. Cool, wet spring weather often
aggravates or induces Zn deficiency in field craps. Zinc deficiency

occurs in calcareous soils with pH of 7.4 or higher because the

solubility of Zn decreases as soil pH increases. In calcareous soil,
adsorption of Zn by carbonates contributes to low Zn availability
(Lindsay, 1972b; and Lucas and Knezek, 1972). Elgabaly and Jenne (1943)
and Elgabaly (1950) reported that some of the Zn2+ and Zn(OH)'*' adsorbed
on montmorillonite was not replaceable and had entered the octahedral
layers of clay. Lindsay (1972a) noticed severe Zn deficiency in corn
planted after beets (high in carbohydrates for microbial growth and
activity) rather than following corn or other crops. DeRemer and Smith
(1964) observed reduction of available Zn to the succeeding bean crop
when sugar beet taps were plowed down. Zinc deficiencies have been
reported in areas where the topsoil containing organic matter was
removed. Lindsay (1972a) concluded that Zn deficiencies increased
because the exposed subsoil was lower in organic matter and higher in pH
and carbonate than the surface soil.

Zinc Metalloenzymes and Effect of the Heavy Metals on Enzyme Activity in

 

Soil

 

The study of soil enzymatic activity is a promising approach to
the study of biological effects of heavy metal ions on the decomposition
process. These effects may be elucidated in more detail than is
possible with simple measurement of soil respiration. More than 10
different Zn metalloenzymes including proteases, transcarboxylases,
carboxylases, phosphatases and dehydrogenases occur in microorganisms.
The metal Zn may have catalytic, functional or both catalytic-functional
importance in these enzymes. They contain fixed quantities of metal
which are firmly bound as an integral part of the protein molecule

(Tyler, 1980). Zinc plays an important role in protein synthesis; it is

essential for normal activity of both DNA and RNA polymerase (National
Research Council, 1979). Removing Zn from DNA polymerase inhibits its
activity; with Zn the complete functionality will resume. Riordan and
Vallee (1974) and Holmquist and Vallee (1976) reported that both

Bacillus subtilis and Bacillus thermoproteolyticus produce enzyme

 

 

proteases in which the atom Zn (1 Zn atom/mole) serves as a component of
an active site and directly participates in the catalytic process
(hydrolysis of the peptides). The enzyme transcarboxylase produced by

Propionibacterium shermanii contains both Zn and Co (2 Co atoms, 4 Zn

 

atoms/mole) and its catalytic reaction is the carboxylation of pyruvate
(Northrop and Wood, 1969). Zinc may maintain the structure of the
protein enzyme and act as a structural constituent as it does in the

enzyme aspartate transcarbamylase which is produced by Escherichia coli

 

and converts carbamyl phosphate to carbamyl-aspartate (Jacobson and
Stark, 1973 and Riordan, 1977). However, in some enzymes (alcohol

dehydrogenase and alkaline phosphatase) produced by Escherichia coli (Li

 

and Vallee, 1973) and Saccharomyces spp. (Simpson and Vallee, 1968;

 

Anderson et a1., 1976), the metal Zn appears to have both catalytic and
structural importance. Alkaline phosphatase has 4 Zn atoms per mole
with 2 catalytically active and 2 are structure stabilizers. The
specificity of a metal in an enzyme may be so exact that substitution by
some other metal species usually results in an almost complete loss of
enzymatic activity. At the same time the metal ion may activate the
enzymes but metal specificity may be lower and similar ions may

substitute with no loss or only partial loss of activity.

Knowledge of the relative effects of trace elements on urease
activity is important because its substrate, urea, is added to soils as
a synthetic fertilizer and in animal excreta. Tabatabai (1977) and Juma
and Tabatabai (1976) showed that 20 trace elements including Zn and Cd
that are commonly found in sludge samples inhibited the activity of
urease and phosphatase in soils and that their order of effectiveness as
inhibitors depends on the soil. Cadmium is known to exert its toxic
effects by replacing Zn in carboxypeptidase enzyme systems which
catalyze peptide hydrolysis (Mahler and Cordes, 1966). Consequently,
the indirect effect of Cd on glucose metabolism may not have been
noticed until large quantities of Cd were present. A cOpper ion reacts
more readily with sulfhydryl groups than Zn and so it is a considerably
more powerful inhibitor of urease activity than the Zn ion (Hughes et
a1., 1969). This was also found by Tyler (1976) to be the case with
phosphatase enzyme where Cu and Zn pollution reduces the decomposition
rate, phosphatase activity, and phosphorus mineralization rate in the
mor horizon of coniferous forest soil surrounding a brass mill in
Sweden. Price (1970) reported that several dehydrogenases were

sensitive to Zn deficiency in Euglena gracilis, therefore the metabolism

 

of aquatic plants can be influenced markedly by changes in the

availability of Zn. In plants, the main function of Zn is as a

metalloenzyme. Several Zn-requiring dehydrogenases, proteinases and
peptidases have been identified in various organisms, including aquatic

plants such as Euglena gracilus (Price et a1., 1972).

 

Characteristics of Sewage Sludge Containing Zn and Other Heavy Metals

 

Application of sewage sludge on cultivated land surrounding a
population center is economical and solves the disposal problem in a
beneficial way. But recently a concern has been expressed regarding the
introduction of heavy metals into natural biological systems in
conjunction with its application on land. Heavy metals pose a hazard to
the food chain since they can be readily transferred from soil to plants
and up the food chain to animals or humans. The chemical composition of
sewage sludge that results from an industrial or domestic activity is
dependent upon numerous factors including the type of chemical digestion
performed, the extent and nature of industrialization in the sanitary
distrdxn:, and the seasonal variability of sewage entering the treatment
facility. Ikfltial studies on the composition of sewage sludge were
primarily concerned with total amounts of N, P, K and trace elements
essential for plant growth. More recently, the metal content of sewage
sludge has received emphasis because of the toxicity of some metals like
Zn, Cd and Ni to microorganisms, plants, animals and even humans.

Bradford et a1. (1975) characterized the metrOpolitan sewage
sludges and effluents in relation to their total and water extractable
trace elements. They found that the total concentration of trace
elements in sludges and effluents were highly variable depending upon
the source. Concentrations of Zn, Cu, Ni, Co and Cd were consistently
greater in the saturation extracts obtained from sludges than those
obtained from a large sampling of California soils. They attributed the
increased solubility of trace elements in sludge extracts to the

formation of soluble metal chelates.

10

Sommers et a1. (1976) collected sewage sludge samples over a 2
year period from eight Indiana cities and analyzed them for C, N, P, K,
Ca, Mg, Fe, Cd, Zn, Cu, Ni and Pb. The dried sludge contained
approximately 50% organic matter and l-4Z inorganic C. Inorganic N,
organic P, K and all metals were found to be quite variable over time
for sewage sludge produced at a given city. Page (1974) mentioned that
Zn concentrations in sewage sludge ranged from 700 to 49 000 mgkg‘l,
whereas Ni ranged from 20 to 5 300 mgkg‘l, Cu from 200 to 8 000 mgkg'l
and Cd from 1 to l 500 mgkg’l. The rationale commonly used to recommend
the rate of sludge application to agricultural land is based upon
limiting available N to that which a crOp can utilize and limiting metal
additions to an amount such that excessive metals will not accumulate in
or be toxic to the crop. The variable nature of inorganic N and metal
contents of sludges indicates that a comprehensive sampling and analysis
program is essential prior to formulating recommendations for rates of
sewage sludge application on soils used for crap production. There is
an inherent heterogeneity in sewage sludges. If one wishes to study the
effect of a particular heavy metal like Zn or Cd in sewage sludge on
microorganisms or soil accumulation and plant response, it is desirable
to have only one kind of sewage sludge and then amend it to get various
levels of Zn rather than having different sludges with varying
concentrations of this metal. Kirkham (1975) added 14 or 140 mgkg'1 Cd
as CdSO4 to an anaerobically digested sludge containing 65 mgkg'1 Cd to
study the uptake of Cd and Zn from sludge by barley plants grown under
four different irrigation regimes. However, metals supplied as

inorganic salts are usually more biologically active than those forms

11

associated with sludge (Cunningham et a1., 1975). Dijkshoorn and Lampe
(1975) found that an application of sewage sludge IflJfll in metals
resulted in about half as much Zn and Cd in potted perennial ryegrass as
when the same amount of the metal was added in a sulfate form. This
variation in uptake may be attributed to the differences in the chemical
form of Zn and Cd in the sewage sludge. Interestingly enough, Giordano
and Mortvedt (1976) found that mobility of heavy metals, including Cd,
from inorganic sources was slightly greater than from sewage sludge.
Sludge organisms can become adapted to toxic substances when these are
continuously present in the waste (Tomlinson et a1., 1966). The
concentration of metals necessary to inhibit nitrification in activated
sludge is much higher than that required to give the same effect in pure
cultures. Tomlinson et al. (1966) found that as much as 150 mgL’l Cu,
added as cupric sulphate, were required to produce 75% inhibition in
unadapted activated sludge plus sewage compared with only 4 mgL'l Cu in
the pure culture. When sewage was replaced by an ammonium salt
solution, nitrification was inhibited at a much lower concentration of
metals suggesting that organic matter removed the toxic metals by
chelation or precipitation. Premi and Cornfield (1969) reported that
nitrification was unaffected, partially inhibited, and totally inhibited
by 100, 1 000, and 10 000 mgkg'l zn respectively. Wilson (1977)
confirmed that nitrification in soil is temporarily inhibited by 100
mgkg'l Zn and totally by 1 000 mgkg-l Zn.

In addition to the chemical and biological changes which would
take place in the land as the result of the sludge application, there

would be changes in the physical structure of the soil as well.

12

Microbial Activity as Affected by Environmental and Heavy Metal

 

Contamination

 

Microorganisms are key components in the biochemical cycling of
various chemical elements, in the incorporation of energy (photo and
chemosynthesis), and in those processes necessary for the maintenance of
the fertility of aquatic and terrestrial habitats and for waste
reduction. Consequently, the elimination, diminution, or enhancement of
any specific microbial pOpulation may influence the overall ecology of
the biosphere. In addition, pollutants may affect complex microbial
interactions, such as parasitism and mutualism (Babich and Stotzky,
1980). Abiotic factors such as pH, Eh, moisture, clay minerals, hydrous
metal oxides, organic matter, ion exchange capacity, solar radiation,
hydrostatic pressure, and inorganic, anionic, and cationic composition
all influence the chemical reaction and hence, availability and toxicity
of the contaminants to microorganisms. Biotic factors like slime
production, physiological age, morphological state, nutritional state,
physiological and genetic adaptation influence the response of the
indigenous microbial pOpulations to pollutants in the environment
(Babich and Stotzky, 1980). The activities, ecologies and population
dynamics of microorganisms in many habitats are influenced, usually
adversely, by the increased deposition of contaminants from both
industrial and domestic sources.

Pollutants are seldom, if ever, present in the environment as
single constituents. Usually, industrial and domestic activities
simultaneously emit numerous pollutants that may be deposited into a

common microbial habitat. Interaction between pollutants, whether

13

synergistic or antagonistic, may evoke responses from the indigenous
microbiota that are different from those evoked by exposure to
individual pollutants.

Most studies of environmental toxicology dealing with
microorganisms have been concerned only with determining the
concentration of a pollutant that causes an inhibitory and/or lethal
response in the target microbe under artificial laboratory conditions.
However, there is little information on antagonistic mechanisms whereby
one heavy metal increases or decreases the toxicity of a second heavy
metal. Antagonistic interactions may result from competition between
metals for common sites on the cell surface with the more efficient
competitor preventing the uptake of another metal. For example,
increasing the concentration of Mg2+ reduced the toxicity of Zn and Cd

to the growth of Aspergillus niger (MacLeod and Snell, 1950 and Laborey

 

and Lavollay, 1973). The uptake of Cd by Euglena gracilis was greater

 

in Zn-deficient than in a Zn-sufficient medium (Nakano et a1., 1978).
However, other studies have shown that although Fe reduced the amount of

inhibition of growth of Aspergillus niger by Cu, Ni or Zn, it did not

 

suppress uptake of these metals (Adiga et a1., 1962). Contradictory
results have been reported by many researchers regarding synergistic or
antagonistic effects of metals and microbial response. Upitis et a1.
(1973) stated that the toxicity of Cd to growth of a Chlorella _s_p. was
reduced by the addition of Zn, Mn or Fe while Hart and Scaife (1977)
reported that Zn addition did not influence the uptake of Cd by

Chlorella pyrenoidosa. No synergistic or antagonistic effect between

 

Cu and Zn was noted in the growth of Selanastrum capricornutum (Bartlett

 

14

et al., 1974). The generation time of a Zn-deficient medium containing
20 mgkg’1Cd was 56 hours, whereas after the addition of 2 mgkg'l Zn, the
generation time was reduced to 27 hours (Nakano et a1., 1978).

The protective effect of one cation on the toxicity of a second
heavy metal cation probably is a reflection of competition for uptake
with a reduction in the subsequent accumulation of the heavy metal
inside the cell. In natural environments, heavy metal pollutants and
the natural inorganic cations each with different chemical affinities
for functional ionogenic groups on the surface of indigenous cells
probably compete in sorption to the cells (Stotzky and Marshall, 1976).

Pre-exposure of Esherichia coli or Saccharomyces cerevisiae to Zn

 

 

permitted the cells to accommodate Cd to toxic levels (Mitra et a1.,
1975 and Macara, 1978). Conversely, low levels of Zn increased the

lethal action of Cd to Klebsiella pneumonia (Pickett and Dean, 1976).

 

Living organic matter (viable cells) sorb heavy metals. They bind
differentially to specific ionogenic groups on the surfaces of these

cells. The cell walls of Saccharomyces cerevisiae have been shown to

 

sorb Cd (Itoh et a1., 1975) and those of Salmonella enteritidis sorb Zn,

 

Mn and Fe by cation exchange (Chipley and Edwards, 1972). Zwarum and

Thomas (1973) reported that cells of Pseudomonas stutzeri have a CEC of

 

3400 Mmol(-+-).kg’l of dry weight and are capable of sorbing substantial
quantities of Al to their cell walls. In the environment, both dead and
living organic matter probably compete for heavy metal ions. For

example, both river sediment and Pseudomonas fluorescens sorbed Hg with

 

the bacterium being the more efficient adsorber (Ramamoorthy et a1.,

1977). However, sediments may sequester sufficient quantities of heavy

15

metals to reduce the effective concentration (availability to the biota)
of the pollutant in that environment. The protective effect of the
sediment was related to its high organic matter content which apparently
removed the heavy metal from solution and thus reduced its availability
to the bacteria (Hamdy and Wheeler, 1978). Soluble organic matter
reacts with heavy metal by either covalent or coordination (chelation)
bonding, and such complexes are, in general, less toxic to the
microbiota than are the free metal ions (Pan et a1., 1961). Another
mechanism of antagonism between heavy metals may involve the sorption of
one heavy metal to the amorphous complex of the other heavy metal. For
example, Steeman and Kamp-Nielson (1970) explained that at alkaline pH
levels, addition of Fe suppressed the inhibition of growth of Chlorella

pyrenoidosa caused by Cu, presumably because of the cupric cations

 

sorbed to the negatively charged sites on the micelles of amorphous
Fe(0H)3, rendering Cu unavailable for uptake by the alga. Synergistic
interactions between heavy metals on microbial cells may result from the
adsorption of both metals to the surface of the cell, with the
adsorption of one metal increasing the permeability to the second.
Although the majority of the responses of microbes to pollutants is
negative, low concentrations of pollutants may, in time, exert a
stimulatory effect on the responding microbiota. According to
Henriksson and daSilva (1978), concentrations of Zn, Cd and Pb ranging
from 0.005 to 0.025 mgkg'1 stimulated nitrogen-fixation of Nostoc 32.,
whereas concentration of these metals between 0.025 to 0.1-5 mgkg'1 was

inhibitory.

l6

Stimulation of the growth and metabolic activities of
microorganisms by low concentrations of a toxicant may reflect an
Arndt-Schulz effect, where the accumulation of a nonlethal concentration
of a poison at the surface of a cell induces, among other changes, an
alteration in the permeability that permits a freer flow of nutrients
across the plasma membrane and, thereby, an increase in cellular
metabolic activity (Babich and Stotzky, 1980). When the concentration
of a contaminant is increased above tolerable levels, the initial
response is an inhibition of microbial activity and, as the
concentration increases, cell death results. Adverse responses of
microbes to pollutants include: prolongation of the generation time of
bacteria; decreased spore production by fungi; decreased nitrogen
fixation by blue-green alga and lichens; decreased photosynthetic
activity of green alga and lichens; and inactivation of viral
infectivity (Babich and Stotzky, 1980).

Babich and Stotzky (1977b) tested a variety of microorganisms for
sensitivity to Cd and noted a wide range. The toxicity of Cd to
eubacteria, actinomycetes and fungi appeared to be pH dependent as it
was generally potentiated at pH 8 or 9. So, it appears that not all
microorganisms are equally susceptible to the toxic effects of metals in
the environment. According to Novick and Roth (1968), strains of

Stafilylococcus aureus carry plasma conferring resistance to Zn, Cd, Hg,

 

Pb and As. Rondo et a1., (1974) also found that the penicillinase

plasmid in Staphylococcus aureus contains gene(s) giving resistance to

 

Cd ions. Resistant cells allow Ca but not Cd to enter the cell. Metal

resistant plasmids are now thought to be common among bacteria, and this

17

could explain the differences found among the species. It is possible
that the cell wall played an important role in protecting the organism.
Tornabene and Edwards (1974) found that when their test bacteria were
exposed to 2 500 mgL‘l Pb, over 99% of the Pb associated with the cells
was on the cell walls and outer membranes of the bacteria. Less than 1%
of the Pb had penetrated into the cytOplasm which may explain why this
extremely high Pb concentration had no effect on the bacteria. Cadmium
would behave in a similar manner to Pb since both cations have many
similar properties.

Fixation and Availability of Heavy Metal by a Soil Component

 

The quantity and availability of the ions in soil are a function
of chemical and biochemical processes alone or in conjunction with other
factors. For example, the pH of the environment into which a pollutant
is deposited may affect the chemical form, mobility, solubility and
toxicity of heavy metal pollutants. According to Lucas and Knezek
(1972), Zn may be rapidly leached under acid conditions from a soil
where rainfall is high. Wear (1956) suggested that as pH of the soil
increases, extractable Zn decreases and there is a reduction in Zn
uptake by the plant. Peech (1947) stated that at a pH of 4.0 virtually
all Zn applied to soil could be extracted, but as the pH increased the
extractable Zn would be diminished. In alkaline environments such as
sea water, Zn exists as a precipitated dibasic compound and Pb as PbOH+
(Hahne and Kroonje, 1973). Cadmium, like Zn, is most mobile in soil at
a pH of 5 or lower (e.g., in acid peat), whereas in soils of high pH
(alkaline soils rich in CaC03), Cd is immobile presumably due to

formation of insoluble Cd(0H)2 and CdC03 (Hutchinson and Collins, 1978).

18

As pH and Eh are related, the ionic form of heavy metal pollution is
influenced jointly by the availability of H+ and electrons in the
environment (Gadd and Griffiths, 1978). KrauskOpf (1956) and Foy et
a1., (1978) reported that under reducing conditions, like those
encountered in anaerobic environments, the microbial conversion of 8042'
to sulfide (SZ‘), may lead to the precipitation of heavy metals such as
ZnS, CdS, HgS and FeS. Adsorption of heavy metal ions may occur on the
surface of Si(OH)4 and A1(OH)2‘ cation exchange sites, and on amorphous
Fe hydroxide below the isoelectric pH. Clay minerals may fix
appreciable quantities of metals by precipitation, by physical
entrapment in clay lattices of metals, and by exchange adsorption (Reddy
and Perkins, 1974). The metal toxicants introduced into the environment
may be adsorbed or exchanged for the cations on the exchange sites of
clay minerals and thereby removed from solution, at least temporarily,
and be unavailable for uptake by the microorganisms. Montmorillonite is
an effective adsorbent of Zn, Cu, Co, Ni, Hg, Pb and Cd, whereas
kaolinite is a comparatively poor exchanger for heavy metals due to its
lower CEC (Babich and Stotzky, 1980).

Increasing the concentration of the clay minerals increased their
protection and this can be related to the CEC, since at equivalent
concentrations, the sequence of protection is in the order of the CEC
magnitudes. However, the protection provided by kaolinite and
montmorillonite for bacteria and fungi in soil against the toxicity of
heavy metals like Cd2+ may not be operable in sea water as Cd exists
there primarily as CdClz and CdCl3' which may not bind as effectively to

the negatively charged clay minerals (Pan et a1., 1961). The sorbtion

19

of some heavy metals by clay minerals appears to be dependent on the pH
of the environment. For example, the sorption of Zn and Pb by
montmorillonite, illite and kaolinite increased when the pH was
increased from 3 to 7; at higher pH values, they were precipitated as
sparingly 011‘ species. This increase was attributed to the adsorption
of OH" groups to the clay, which presumably acted as bridges to metal
ions or through abstraction of a proton from functional groups. The
ability of the clay minerals to exchange heavy metals is apparently also
influenced by the concentration and type of soluble ligands present in
the various environments. For example, in sea water, both OH“ and Cl‘
anions compete for the same metals: with Zn and Pb, PbOH‘" and Zn(OH)2
(which is sparingly soluble) species predominate over Cl' complexes.
The form and chemical activity of many heavy metal pollutants is not
only influenced by the concentration and type of the anionic ligand
present in the environment, but these different coordination complexes
exert different toxicities to microorganisms. Microorganisms produce
biochemicals such as amino acids, sugar acids, phenols, simple
aliphatics and other compounds in soil which chelate metal ions.
Although these constituents have only a transitory existence,
significant amounts may be present in the soil during periods of intense
biological activity which occur near decomposing residues, in the
rhizosphere and in the soils amended with organic wastes (Stevenson,
1982). Biochemicals produced in this way as well as other chelating
compounds occurring naturally in the soil (humic and fulvic acids) are
all rich in functional groups (metal binding sites) and have the

capability of transforming the solid phase forms of metals into soluble

20

and/or stable complexes. Even though simple biochemical compounds are
of little significance in soil because of their rapid destruction by
microorganisms, in many agricultural soils with high organic matter
content (0M) the combined total of potential chelate formers in the
aqueous phase is probably sufficient to account for the minute
quantities of metal ions normally present (Stevenson, 1982). Zinc, like
other multivalent cations (Cu+2, Mn+2), has the potential for complex
formation or linkage with humic acid. Randhawa and Broadbent (1965)
reported that in the retention of Zn by humic acids, three types of
sites are involved. The less stable complexes were believed to be
associated with phenolic OH groups and weakly acidic COOH; the more
stable complex was believed to involve strongly acidic COOH. Although
the strongly bound Zn represented less than 1% of the total retained,
the sites responsible were believed to be of great importance because
small quantities of Zn would be bound preferentially as the most stable
complex. Humic and fulvic acids form both soluble and insoluble
complexes with polyvalent cations depending upon degree of saturation.
However, metal complexes of fulvic acids are more soluble than those of
humic acids because of their high acidities and relatively low molecular
weights. According to Stevenson (1976, 1977) the metal complexes are
soluble at low metal-humic acid ratios, but precipitation occurs as the
chainlike structure grows and COOH groups become neutralized through
salt bridges.

Stevenson (1982) reported that in the natural soil a balance
exists between the metal ions that occur in the solution phase (free

ions and/or soluble chelate complexes) and in insoluble mineral and

21

organic forms.

The quantity and availability of micronutrients at any one time
are affected by the synthesis and destruction of biochemical chelating
substances as well as by transformations carried out by microorganisms.
The requirement of micronutrients for bacteria, actinomycetes, and fungi
are the same as higher plants, and they immobilize available nutrients
when levels are subOptimum for growth. The relationship is analogous to
N immobilization when crop residues with wide C/N ratios undergo
decomposition in soil. According to Tisdale and Nelson (1975) when a
C/N ratio in OM is greater than 30, there is immobilization of soil N
during the initial decomposition process. For ratios between 20 and 30
there may be neither immobilization nor release of mineral N. And if
the C/N ratio is less than 20, then there is a release of mineral N
early in the decomposition. Besides the C/N ratio, many other factors
influence the decomposition of organic materials and the release or
immobilization of N.

Effects of Sterilization on Soil and Microbial Response

 

Sterilization is the process of killing or removing all living
cells from a medium. It can be accomplished by exposure to lethal
physical or chemical agents or, in special cases, by filtration of
solutions. The maintenance of cellular equilibria is a function of
various enzymes which in turn are regulated by genes on the cell
DNA-chromosome. Many essential enzymes are associated with the
cytoplasmic membrane which must be intact in order for the enzyme
activities to contribute to cell maintenance (Carpenter, 1972). The

vital activities of any organism or cell helps to maintain a dynamic

22

state or equilibrium condition. The disturbance of any cellular
equilibrium by altering the factors controlling it may lead to cellular
death. Radiation can induce in the medium reactive chemical radicals,
of which the most important is the hydroxyl free radical (OH). It is
the most reactive and potent oxidizing agent of various oxygen
intermediates known and is capable of attacking many of the organic
substances present in cells (Brock, 1979). Ionizing radiation can act
on all cellular constituents, but death usually results from effects on
DNA. The only valid criterion of death in the case of microorganisms is
irreversible loss of its ability to reproduce. This is usually
determined by quantitative plating methods with survivors being detected
by colony formation. Cell membranes may be altered in permeability, be
dissolved, be removed or be ruptured by sterilization and therefore
cause destruction of the equilibria that maintained constant composition
and osmotic pressure. Increased membrane permeability would permit
toxic substances to enter or vital components to be lost from the cell
and decreased permeability would retard the entrance of nutrients or
excretion of toxic wastes. In studies of the chemistry and microbiology
of soil, it is often desirable to work with a soil which is sterile, but
use of heat or chemicals for sterilization changes the chemical and
nutritional prOperties of soils rather markedly (Russell and Russell,
1950). The use of X and X—radiation or an electron beam provides a
useful means for partial or complete heatless sterilization of soil with
the possibility of leaving the soil and its enzymes largely unchanged
(Paul and McLaren, 1981). RadioisotOpes in general, and especially

those which emit x-rays, provide high energy radiation which far exceeds

23

many chemical bond energies and can induce chemical reactions and/or
disrupt structures in biological systems. Ionizing radiation occurs as
a result of Spontaneous disintegrations or transformation of atomic
nuclei. These nuclear changes can give rise to several types of
radiation; one beingX-rays which have an electromagnetic nature
(photons) (Wolf, 1969). Photons are electrically neutral particles with
zero mass when the particle is at rest and, consequently, they can
penetrate matter with little interaction. This characteristic allows
X-rays to have a greater effective range in matter than that of alpha or
beta particles of comparable energy (Wang et a1., 1975). Gamma
radiation causes ionization, and brings about sudden and drastic changes
in the microbial population of soils. McLaren et a1., (1957) sterilized
the soil by an electron beam of sufficient intensity and energy and
showed that doses of l and 2 million roentgen (roetgen(r) = 98 ergs g'1
of tissue) will kill all fungi; and eliminate all bacteria and
actinomycetes respectively in soil. At a dose of 2.4x105 r the number
of viable organisms are reduced to less than 17.. They also reported
that soil urease activity was not retained in a similar soil where
autoclaving was used as a means of sterilization. Doses of 2, 8, 32,
64, 128 and 250 kiloroentgens (kr) of gamma rays from a Go60 source were
used by Stotzky and Mortenson (1959) to irradiate samples of Rifle peat.
No significant effect was observed 54 days after an incubation period on
the development of the bacterial population, pH, mineralization of
phosphorus and evolution of C02. Growth of the fungal population and
ammonia utilization was increasingly inhibited by doses of 8 to 250 kr.

Since gross changes did not take place in the metabolism of the soil

24

Inicroflora, apparently higher dose levels are required than those used
for the sterilization of organic soils. Arredondo fine sand was exposed
to gamma radiation from a C060 source at doses of l, 4, 16, 32, 64, 256,
512, 1 024 and 2 048 kr. Popenoe and Eno (1962) reported that the
percentage survival of fungi and bacteria decreased with each increase
in radiation dose to 4% at l 024 kr. Algae were not as drastically
reduced as bacteria and fungi. Some survived at 2 048 kr but greatest
reduction occurred above 64 kr. With 1 024 kr, a few nematodes remained
in the soil 2 days after irradiation, but at a dose rate¢fl5256 kr,
after 14 and 28 days none were recoverable. Two populations, one more
sensitive to radiation than the other, were probably involved in this
reaction. Stotzky and Mortensen (1959) reported that the number of
viable cells diminished as a function of radiation dose and that fungi
werelmnxasusceptible to radiation damage than bacteria in the pure
culture. McLaren and et a1. (1957) stated that bacteria and bacterial
spores are more sensitive to electron beam radiation than viruses and
enzymes. 1hr: example, bacteria and their spores require from 2x105 to
2x106 r for 100% sterilization in broth, whereas urease in soy flour was
only inactivated 9.4% by 2x106 r. These results also indicate that
comparable doses are required for killing microorganisms in soil. Comar
(1955) reported examples of physiological response in X or Y-radiation
of many organisms. He indicated that dosages of 200 000 r, 500 000 r,
and 1 000 000 r destroyed bacterial colonies, sterilized spores of
bacteria and inactivated tobacco mosiac viruses, respectively-
According to Carpenter (1972), the ionizing radiation can produce

mutants in the surviving population besides the death which it causes by

25

rupturing the cell surface. These genetically altered forms differ from
the parent p0pulation usually in only one characteristic, such as
resistance to bacteriophage or the ability to synthesize a substance
necessary for metabolic activity.

Bacteria vary markedly in sensitivity to ionizing radiation

(Brock, 1979). Typical sensitive organisms are species of Pseudomonas,

 

and resistant organisms are species of Micrococcus and Streptococcus.

 

 

Probably the main reason that spores are resistant is their low water
content, which reduces the efficiency of ionization events. Another
factor affecting radiation resistance is the presence of chemical
protective agents. These are mainly organic sulphydryl cunnpounds that
react with radiation-induced ionized substances converting them to
normal substances. Such protective compounds may be present in high
amounts in organisms that are unusually radiation resistant.

There is little information concerning the influence of soil
organic matter on the lethality of radiation toward the microflora,
although work of Dertinger and Jung (1970) with organisms in pure
culture indicated that some organic compounds of high molecular weight
have a protective effect that may be partly explained by their
reactivity with free radicals.

Use of Soil Perfusion Apparatus in Studying Microbial Processes in the

 

Soil

lees and Audus first developed the soil perfusion apparatus in
1946. Temple (1951) described a modified design of the Lees's device as
being a useful tool for the study of microbial processes and

investigation of soil reactions. This modified and compact unit has the

26

advantages of seldom requiring mechanical adjustment, having fewer glass
to rubber joints and being easier to maintain in aseptic or
uncontaminated conditions. The perfusion technique is applicable to
many bacterial systems utilizing a water-insoluble substrate and is
superior for static studies of many processes in the soil such as
chemical and biochemical metal fixation, nitrification and pesticide
degradation where a solid is immersed in a liquid medium. The apparatus
was especially well adapted to the studying of bacteria on sulfur
compounds in coal and associated rocks. Substances other than soil,
coal or rock also may be used in the unit. For example, Thiobacillus

thiooxidans grows well in this unit on Waksman's sulfur medium when the

 

sulfur is placed in the inner perfusion tube. Wildung et a1., (1969)
reported that perfusion units were useful in investigations designed to
establish rate and mechanisms of pesticide degradation, in isolating
organisms responsible for this process and evaluating the role of
environmental variables on rates and pathways of degradation. The
precise control of soil moisture and soil atmosphere composition during
perfusion was accomplished by regulation of pressure and composition of
ggases surrounding the soil column. However, due to a lack of uniformity
Of moisture and aeration regimes within the soil column, the
relationship between degradation rates in perfusion units and field
Condition was difficult to evaluate. Stewart et a1. (1975) used soil
perfusion techniques to study the transformations of N in fractions of
anaerobically digested sewage sludge. The soil perfusion experiments
1tidicated that only the ammonium-N of the sludge solids was oxidized to

IVO3-N and the rates of nitrification of sludge were 37 ug No-3'-N/g soil

27

day '51 for 2.5 cm hr’l. Zamani et a1. (1980) utilized a soil perfusion
system and extensively studied static Zn immobilization by
microorganisms as well as organic matter in different soils. The system
'was structured to resemble the natural soil system where the solid phase
(soil body) was immersed in a liquid medium (application of liquid
sewage<m1the land). Contamination of underground water (medium in
filter flask of the soil perfusion apparatus) was possible if the soil
and its enhanced microbial pOpulations did not act as an effective
"living filter".

The ability to directly sample at any specified time interval and
analyze the solution which percolated through the soil was a major

advantage of the soil perfusion system over other methods.

MATERIALS

.8231:

Surface samples of Rubicon sand (Muskegon County, MI) Brookston
clay loam, (Ingham County, MI) and Houghton muck (Clinton County, M1)
were used to compare the relative amounts of Zn inactivation in soil due
to microbial activity and to physical and chemical factors. The Rubicon
sand and Brookston clay loam were air dried, passed through a 2 mm sieve
and stored in the air dry state at room temperature (25°C) until
required for experimental work. Houghton muck was passed through a 2 mm
stainless steel sieve and stored in a moiststate to avoid rewetting
problems.

Soil Perfusion System

 

Soil perfusion units of a modified design of the Lees soil
percolation apparatus (Temple, 1951), as shown in Figure l, were used in
the soil perfusion experiments. Soil perfusion units were connected to
a vacuum pump in series with the particular experiment being conducted
determining the number of soil perfusion units in use (Figure 2).

The soil perfusion unit is a vacuum driven device which can be
used to move perfusate from a soil perfusion reservoir through a soil
column under a variety of experimental conditions such as aseptic
environment, type of soil column, composition of perfusate,‘and aerobic
or anaerobic conditions. Soil perfusion units are well suited for
experiments which require accelerated impacts of particular soil
microorganisms upon the biology and chemistry of soils under specific

environmental and experimental conditions. The perfusion cell or unit

28

29

kumowvcH msu muoz

.Hfiom cmxoma Mo :EDHoo

.mmflvsuw mmmnu CH pom: mzumumaam cowmswuma Hwom

.H muswwm

30

 

31

kucwu memomn mmmau

.cowumfiuwu mesmw mo uasmmu m we :Boun

.mmfiumm ca umuooccoo mufic: conswuma HHow mounH

.N muamwm

32

 

33

is composed of two pieces of pyrex glassware. One part is a 500 mL
reservoir or filtering flask with a stOppered cap and a built-in needle
for sample removal. Air entry ports are part of the reservoir design so
that a constant supply of fresh filtered air is available to the cell in
experiments under aerobic conditions. The second part of the soil
perfusion cell is the sample percolation unit constructed of Pyrex
tubing of an appropriate size to allow a vacuum driven device to
function.

Soil Perfusion Column and Perfusate

 

The soil perfusion soil column was prepared by combining 12.5 g of
soil with 12.5 g of silica sand and l g of glass wool. A plug of glass
wool was inserted into the bottom of the soil perfusion column, and the
mixture was inserted into the soil column tube (Figure l) and packed 111
such a manner that a uniform rate of percolation and distribution of
perfusate was obtained regardless of the soil being used or experimental
treatment being applied. The column and all other openings of the soil
perfusion system were sealed and the soil perfusion unit was wrapped
with aluminum foil to reduce recontaminathnicfi theIHUt after
sterilization by either autoclaving or by gamma irradiation using a
'variable flux gamma irradiator with a 60C0 gamma source. Filter flasks
containing 320 mL of deionized water to be used as perfusate were capped
‘with steppers containing needle-entry ports, covered with aluminum foil
and subjected to the same sterilization procedure. The Zn solutixni and
microbial energy and nutrient additions were subjected to similar

sterilization procedures.

34

Energy and Nutrient Enrichment of Perfusate

 

Denatured Difco bacto nutrient broth solution was selected as the
energy and nutrient supplement used to enhance microbial activity in the
soil perfusion systeuu The five mL solution added 389 mg of C and 140
mg of N (C:N ratio of 2.8:1) to the 320 mL of purfusate in the
reservoir. A 32 hour growth period was required after an energy and
nutrient addition and microbial inoculation before maximum microbial
activity was achieved and experimentation with Zn addition could begin.

Agar Plate Preparation for Dilution and Counting

 

A mixture of 1 kg soil, 1L H20 and 10 g CaC03 were autoclaved for
one hour, cooled and filtered through Whatman #2 filter paper. The soil
extract was adjusted to pH 7 and sterilized. A.subsample of 200 mL was
mixed with.800 mL H20, 1 g glucose, 15 g dipotassium phosphate and 15 g
bacto-agar. The pH was again adjusted to 7 and the mixture autoclaved
for 15-20 minutes and then either mixed with labeled Zn, unlabeled Zn
source, or unaltered and poured into dispo petri«dishes and allowed to

solidify.

METHODS

8011 Characteristics and Textural Analysis

 

Rubicon sand soil (Muskegon County, MI) was used in all the soil
perfusion experiments. Brookston clay loam (Ingham County, MI) and
Houghton muck (Clinton County, MI) were also used in selected
experiments. The methods and procedures used in the chemical and
physical characterization of the soils are those employed in the
Michigan State University Soil Testing and Plant Analysis Laboratory
(Dahnke, 1980). The chemical and physical properties of these soils are

presented in Tables 1 and 2.

Table 1. Textural analysis of Rubicon sand and Brookston clay loam.

 

Particle size
Soil series

 

 

 

 

sand silt; clay

%
Rubicon sand 88.5 4.4 7.1
Brookston 22.5 32.4 35.1

clay loam

 

Inorganic Zn Analysis

 

A one mL sample of the soil perfusate was removed from the
perfusion reservoir at periodic intervals during the operation of the
soil perfusion system and brought up to a 10 mL volume with deionized

water. Analysis was conducted using a Perkin Elmer-303 atomic

35

36

.mcofiumofiaamu sauna mo mwmum>m mzu mum mmDHm> HH<+

 

 

 

 

 

 

 

x056

«Om ma cm a oa.~ Nm RN 0 mm mom comm ooa on 0.0 scunmsom

smoa mmao

mna mm ON n ON. 00 «a mo Ha mmq oqmm wee me m.~ coumxooum

vamm

mm «a «m N ma. ON H m e mm omm ca Ha m.m cooansm
lhlwxeA+vHOEE N HIE m6 llllllll UNI wx ma IIII I llllll Hwa ma lllll III

omo w: mu M on so :2 :N ms nu x m ma mmfiumm

momma wuamm
wanmomcmnoxm mansaom manmuomuuxm do: 2 H.o mucmauusc manmuomuuxm Haom
+.xo=a acunwsom was amoa mmao coumxooum .vamm cooflnsm mo moaumwumuowumno Hwowaono .m manna

37

absorption spectrophotometer.

Inhibitory (static) and Lethal (cidal) Effect of Zn

 

Duplicate samples of 0, 10, 20, 30, 40, 50, 100, 150, 200, 250 and
300 mgkg"1 Zn in a 40% volume of thioglycollate medium were autoclaved
for 20 minutes, cooled, aseptically inoculated with 0.1 mL of soil
suspension (1:1 soil:water) and incubated for l to 2 weeks at room
temperature.

Folhndnglflm incubation period, those samples showing no
microbial growth, as indicated by turbidity, were subsampled ixnua fresh
thioglycollate medium, set aside for 1 or 2 weeks at room temperature
and then checked for microbial growth. If microbial growth was nil, a
cidal effect from zn on microorganisms was assumed.

Sterilization and Microbial Growth Inhibitors

 

Autoclaving the intact perfusion unit (with packed soil column in
place) for one hour per day on 2 consecutive days and for 17 hours on
the third day at 120°C at 1.1 kg cm‘2 was found in several trials to be
the most effective means of steam sterilization and was used in all
subsequent autoclaving experiments. Similarly, the most effective
sterilization using gamma irradiation was accomplished by 6000
irradiation for 24 hours with a total radiation dosage of 4.9 megarads
of the entire soil perfusion unit with the packed soil column in place.

Tb insure uniformity of sample treatment, all treatments were
autoclaved or irradiated, and then those samples scheduled to contain
microbial activity were reinoculated with a fresh soil suspension (1 g
unsterilized soil:10 mL deionized water) made up from the soil being

studied. A similar soil suspension, which had been sterilized, was

38

added aseptically to the treatments scheduled to have an inactive
microbial population so that a complete check could be maintained.

A.microbial viability test was performed before and after each
sterilization and the sterilized perfusate was monitored throughout the
experiment as regular aseptic perfusate withdrawal raised the risk of
contamination. To determine the effectiveness of the sterilization and
subsequent aseptic manipulations, one mL of soil perfusate was added to
10 to 15 mL of sterile bacto-fluid thioglycollate medium and was
incubated for one to two weeks at room temperature. After preparation,
the thioglycollate medium separated into two colored layers, the pink or
aerobic zone on the top (containing resazuring as an Eh indicator) and
the yellow or anaerobic zone at the bottom. If the incubated solution
appeared turbid in either zone, microbial activity was present and
complete sterilization had not been achieved.

Uniformity of Perfusate Solution Mixing

 

The soil perfusion units were packed with identical mixtures of
soil, sand, and glass wool and perfused with 320 mL of solution
containing 20 mgkg‘l Zn (as ZnClZ). Addition of a few drops of a dye
mixture of bromo-cresol and methyred (an indicator for N determination)
to the soil perfusion system while it was in Operation indicated a
complete mixing action while the Zn contents of drawn samples from
different units were compared to verify uniformity between units.

8011 Perfusion System Operation in Experimental Mode

 

The sterilized soil perfusion units were assembled in a series and
hooked to a vacuum pump as shown in Figure 2. Either 5 mL of soil

suspension and 5 mL of sterile deionized H20 or 51m3cfi’sterile soil

39

suspension plus 5 mL of nutrient broth were injected into a column and
incubated for 32 hours to allow for microbial growth. Following
incubation, the soil perfusion units were started and regulated. A
‘baseline soil perfusate sample was drawn and five mL of l 000 mgkg"1 Zn
(5 000 ug Zn/320 mL perfusate) solution was added to the soil perfusion
units by aseptic injection onto the soil column. Eleven serial samples
were drawn over a period of 72 hours and analyzed for microbial activity
and Zn content. The total volume of the system was kept constant by
timely additions of deionized H20.

Statistical Procedures

 

Facilities of the university computer center and STAT’routines
system (version 4.4) of the Agricultural Experiment Station were used
for statistical analysis. The perfusate Zn concentration data were
subjected to an analysis of variance. The experiment was arranged in a
split plot design with 3 replications. The main plot was treatment
which consisted of four combinations of inoculation with microorganisms
and amendment with nutrient broth:

sterile and unamended

sterile and amended

inoculated and unamended

iooculated and amended
The sub-plot was time of sampling which occurred at 12 time intervals:
0, 0.5, l, 2, 4, 8, 12, 24, 36, 48, 60 and 72 hours. Simple effects of
treatment and time of sampling and the interaction between the two were

carefully analyzed.

40

Dilution and Counting of Soil Microorganisms

 

The Rubicon sand soil perfusion experiment ran 72 hours during
which the maximum number of Zn immobiliZing-microorganisms developed.
One g of soil from the soil perfusion column was mixed with 10 mL of
sterile deionized water. Dilutions of 1:10n (where n varied from 1 to
8) were prepared in a sequential manner by transferring 1 mL of a
previous dilution into a 10 mL volumetric flask and filling it to volume
with sterile deionized water.

In order to determine which dilution gave the desired number of
colonies, replicate plates were prepared by inoculating 0.1 mL from each
of several dilutions in the anticipated critical range. The diluted
soil suspension was placed on the agar plate medium and spread in a
manner that resulted in a good distribution of organisms. The plates
were then incubated at room temperature until the colonies became
visible. The assumption is that each viable microorganism trapped in or
on a nutrient agar medium would multiply and produce a visible colony.
The number of colonies would therefore be the same as the number of
viable microorganisms inoculated on the agar in the petri dish. Plates
with yields between 30 and 300 colonies could be counted easily and
accurately.

Isolation of Zn Immobilizing Soil Microorganisms

 

A technique combining standard microbial plating techniques in a
65Zn impregnated agar and autoradiography was devised to determine what
percentage of the microorganisms in the soil pOpulation were
immobilizing Zn. The technique was utilized to isolate pure cultures of

the Zn immobilizing soil microorganisms.

41

Tracer Zn solutions of 0, 11 000, 22 000, 33 000, 44 000, 66 000,
88 000, and 176 000 disintegrations per second, equivalent to 0.0,
0.024, 0.048, 0.072, 0.096, 0.144, 0.192 and 0.384 mgkg‘l 652n, were
mixed with soil agar and the extract was transferred to petri dishes and
allowed to solidify. A separate set of agar plates with non-labeled Zn
in identical concentrations was also prepared to determine the toxicity
of ionizing radiation. Both sets were spread with diluted soil
suspension and then incubated for 4 to 7 days. Response of x-ray film
to the time of exposure was determined for each concentration of
radioactive Zn by selecting the plates with a specific concentration
which had a desired number of colonies (30 to 80), setting petri dishes
above the film and exposing KODAK NO-SCREEN X-RAY film (NS-2T) or
X-OMAR-AR film for various time periods. A good response consisted of
distinct black spots on the processed film relating directly to 652n
concentration and length of exposure time to the film.

Isolation of Pure Culture of Microorganisms

 

The microbial biomass (colony) uhich accumulated radioactive 65Zn
on the agar plate was marked carefully by using a glow box (source of
light) to match with the distinct exposure spots on the films.
Overlapping colonies and contamination of different species of
microorganisms on plates as the result of overgrowth during lengthy
periods of x-ray film exposure time occurred so doses of 652n were
adjusted to speed up the autoradiograph. The use of the streak plate
technique with subsequent transfer to a tube culture after colonies were
formed provided a simple procedure for isolation purposes. A transfer

needle was sterilized and inserted into a specified colony and a loopful

42

of inoculum was streaked rapidly and lightly back and forth across the
new medium. Under sterile conditions, the plate contained a single
group of Zn immobilizing microorganisms.

After colonies developed, a 1o0pfu1 of colony was transferred to a
new culture tube and stored in a cold room until time for
identification.

Identification of Zn Immobilizing Microorganisms

 

Staining techniques and microsc0pic examination were used to
identify the Zn immobilizing microorganisms. Inoculant was removed from
the refrigerated culture tubes with a sterile loop and placed on the
plates containing special growth media. Potato dextrose agar (3.9 g 100
mL'1 of deionized H20) was used for fungal growth and tryptophan glucose
extract agar (2.4 g 100 mL"1 of deionized H20) was used for bacteria and
actinomycetes. New colonies developed on the respective media after a
short incubation period of 2 to 4 days and were stained for microsc0pic
study. Staining of cell samples consisted of spreading a drop of an
aqueous cell suspension on a glass slide, allowing it to dry, applying
gentle heat to fix the cells, followed by the.application of the
staining solutions. The cells were stained by the application of a
single staining solution (such as lactophenol cotton blue for fungi).
Differential staining procedures were used to study the bacteria and
actinomycetes as follows: Using a sterile transfer loop, two loopfuls
of water were placed on a clean, dry slide. A portion of a colony was
spread to cover an area of 1.5 cm. It was air dried and then fixed by
flame. After slide preparation, selected stains were applied to the

smear: Crystal violet, one minute; the smear rinsed with Grams iodine

43

and then sustained for an additional period of one minute; rinsed with
95% ethanol and then restained with a few drops of safranin. After 30
seconds excess stain was removed by gentle running water, and then
blotted dry with absorbent paper. A slide stained and prepared in this
way was studied under the light microscope using the oil-immersion
objective.

Identification of an organism is the process of determining its
species. As many as possible of its characteristics are ascertairuui by
.appropriate observations and tests, and the accumulated information was
then compared with published descriptions of the various species. The
organism was considered properly identified when a species description

was found that was identical with the observed characteristics.

RESULTS AND DISCUSSION

The information presented in Tables 1 and 2 show that Rubicon sand
is a relatively infertile sandy soil with a low cation exchange capacity
(32 mmol(+)-kg"1) and a pH of 5.9 while Brookston clay loam is a very
fertile soil with a moderate cation exchange capacity (175 mmol(+)-kg‘1)
and a pH of 7.5. Houghton muck, by contrast, is an organic soil of
moderate fertility, a higher cation exchange capacity (705 mmol(+)°kg‘l)
and a pH of 6.0. The data suggest that Rubicon sand should be an ideal
soil for microbial immobilization of excessive Zn addition provided
nutrients and energy are supplied to the soil to enhance the microbial
population.

Verification of consistent performance between the three soil
perfusion units used in the experiments is given in Figure 3.
Performance consistency between different soil perfusion units was
determined visually by rate of mixing of a dye as well as by analyzing
the Zn concentration in the soil perfusion reservoir of each unit with
time. Uniform distribution of the dye solution after introduction into
each unit indicated a complete mixing action. Similarity of Zn
concentration in the samples drawn from separate units at specified time
intervals verified the operational consistency of multiple units
connected in series (Figure 3). The Zn in the perfusate reached an
equilibrium concentration of about 4 mgkg'1 prior to five hours of
operation.

Effective soil sterilization should reduce Zn removal from the

perfusion solution if microorganisms are involved in the removal. The

44

 

 

45

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mmB ufio>uomwu mzu ca cofiumuucmucoo am one can .oumu Hmsvm Esau um cesaoo
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<1 .muHCD coamSMCma Haom ucmumwwwv mmunu mo zocmumwmcoo mommEuowqu one

.m muswfim

46

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47

data in Figures 4 and 5, respectively, show that autoclaving and
gamma-irradiation suppressed Zn removal from the perfusion solution.
The removal of Zn from Rubicon sand soil perfusion solution after
sterilization, addition of nutrient broth and inoculation with a fresh
microbial pOpulation was similar in both methods. However, the
equilibrium level of Zn in the soil perfusion reservoir sterilized by
gamma radiation (Figure 5) was only half that with autoclaving (Figure
4). This observation indicates that hydrolysis of organic matter may
have occurred during autoclaving and this left more soluble Zn-organic
complexes in the reservoir. Therefore, the Zn level was higher in the
autoclaved perfusion solution than in the gamma-irradiated solution
after sterilization. Based on this information, the relatively
nondestructive gamma-irradiation technique was used for microbial
suppression.

Data in Figure 5 also show that inoculation of a sterile and
amended perfusion treatment after 68 hours of incubation will induce Zn
removal from the perfusion solution. These results are evidence that
the Zn removal pattern observed is indeed due to the presence of soil
microorganisms from the inoculate.

Additional evidence for soil microorganisms being involved was
through filtration of the perfusate. Results of unfiltered and
corresponding filtered samples from soil treated with increasing amounts
of a food and energy source (1, 2 and 3 mL soybean meal extract) as
represented in Table 3 indicate that the Zn concentration was reduced
noticeably by as much as 2.8 mgkg’lzn as the result of filtration or

almost 53%. Since the pore size of Gelman Acrodisc disposable filter

48

assembly (0.2 u) is smaller than the general size range of
microorganisms (0.5 to 20-50 u), it would therefore eliminate the
microorganisms which presumably immobilize Zn. Beside microorganisms,
this screening action would also eliminate large sized Zn-adsorbing
particles (>0.2u) such as colloidal clay, organic matter, and microbial
products or by-products. The filtration method was only indirect
evidence that microorganisms participate in Zn-immobilization in the

soil.

Table 3. Filtration of the soil perfusate over time to determine
if Zn in the reservoir is present in a particulate form.

 

 

Organic Zinc concentration (hrs after addition)
matter
level Filtered 4 15 32 39
(0.2 u) mg kg"1
1 no 5.0 2.3 2.3 1.8
yes 4.7 1.7 1.8 1.2
2 no 6.9 2.8 5.5 4.5
yes 6.5 2.2 3.5 2.4
3 no 600 303 501 503
yes 5.4 2.3 3.7 2.5

 

Zinc removal data for replicated treatments consisting of sterile
and unamended, sterile and amended, inoculated and unamended, and
inoculated and amended soil perfusion systems with Rubicon sand,
Brookston clay loam and Houghton muck soil columns are given.iml Figures

6, 7 and 8, respectively.

 

49

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tam .SHmoflooHumq emamemm mm: uHo>ummou mnu Eoum mumaooumm .wsfi/maoousm
xe vmuwawumum mmB suit... maumumaqm cowmsmumm Hfiom m CH coaumnfiznoaefi

cw com: :uonn ucmfinusc consumcmu pew msmwcmwuoouofie mo mucosamca one

.q muswam

 

50

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uouasa: uogsnpad u!
(,,6)|/6u1)uog;enuaouoa uz

 

51

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oHEOum kg :N pow commamcm cam zaamowvoHumm anaEmm mmB uHo>ummmu mnu Eouw
mumaooumm .cofiumuwafinoaafi :N ca mmmmuocfi ucosvmmnsw m cufis use: Sumo mnu
um ucmeummuu mdnmum mco cu woven v.33 mEmwcmwuoouowz .cowumavmu maamw
an vmnwafiumum was coats maumumaam :ofimswumm HHom w ca cofiumuuawnoaaa

:N com: nuoun ucmfiuusc wmusumcwv new mamacmwuoouoae mo mucosamcfi one

.m wpswam

52

.m 939“.

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tuneup; tea 33230:. O
5:339: .2 mm new caucus; 6:85 B
caucus; new 3.85 Q

 

 

 

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(,.5x/6w) uonenuaouoo uz

53

Data in Figure 6 for Rubicon sand show that the inoculated and
amended treatment resulted in a significant greater removal of Zn from
the soil perfusion solution with time than did the other treatments.
These results imply that inoculation with viable soil microorganisms and
amendment with nutrient broth stimulated microbial growth and subsequent
Zn immobilization. The increase in Zn removal was significantly greater
than the amount removed from solution by the control treatment (sterile
and amended). While not significant, there tended to be a greater Zn
removal with the inoculated and unamended as compared to either sterile
treatments. Therefore, the Rubicon sand appears to need supplemental
nutrients and an energy source to produce enough of a microbial
population to achieve maximum immobilization of Zn. The Zn levels in
the perfusion solution generally reached an equilibrium state within 12
hours even though the inoculated treatments tended to immobilize
additional Zn with time.

Data in Figure 7 for Brookston clay loam show a rapid inactivation
of Zn from the soil perfusion soil to a level less than one mgkg'1 with
no significant difference between treatments. These results indicate
that chemical and physical factors dominated the control of the level of
Zn in the soil perfusion system while microorganisms were relatively
unimportant in the inactivation. Such results were expected since
Brookston clay loam has a pH of 7.5 and a high clay content and cation
exchange capacity to provide surfaces and sites for Zn adsorption.

The information shown on Figure 8 for Zn removed from the soil
perfusion solution with Houghton muck shows no significant difference

between treatments. Presumably, the large number of active organic

 

54

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HHom m CH vaHmucoo CECHoo HHom qum CooHnCm m CH CoHumNHHHnoEEH

CN COCC Cuoun quHuuCC CmuCumCmv CCC mEchmeoouoHe mo wocmCHmCH 0:9

.0 qumHm

55

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JIOMGSOJ uogsnuad u;

(“Bx/Btu) uonenuaouoa uz

 

56

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57

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58

 

 

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59

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60

binding sites enabled the Zn level to be buffered at a one mgkg'l level
regardless of treatment. While the difference in Zn levels in solution
were not statistically significant, there did tend to be a greater
inactivation with the inoculated and amended treatment.

Data in Figure 9 show the results of Mn immobilization in Rubicon
sand. The procedures are the same as those used in obtaining the data
for Zn shown in Figure 6 except that Mn, as MnSO4, rather than Zn was
applied. Results given in Figure 9 showed that microbial activity and
organic amendment significantly reduced the level of Mn in the soil
perfusion solution and the patterns of Mn immobilization with time were
similar to that of Zn. The phenomenon of relatively rapid inactivation
of Mn in this experiment was attributed to either a complexation or
adsorption phenomenon in the soil organic fraction or to enriched
microbial fixation as reported by numerous investigators. The higher
equilibrium values of Mn than Zn in the perfusion solution at the end of
the experiment was probably due to less precipitation and adsorption of
Mn than Zn under these experimental conditions.

The dark areas in Figure 10 represent autoradiographic exposure of
x-ray film by isolated colonies of 65Zn-immobilizing soil microorganisms
isolated from a perfused Rubicon sand column and which were grown on an
agar plate impregnated with 65Zn. Larger exposure areas represent a
greater accumulation of the radioactive isotOpe. The greater 652m
accumulation could be due to either larger colony mass or to a
differential amount of uptake by different microorganisms. Since only
20% of the colonies formed accumulated 652n in amounts discernable from

background levels by autoradiography, there is good circumstantial

 

61

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mumHooCmm .COHumHCmC meamw an woNHHHCmum was :oHF3 mCumumCCm ConCwumC
HHom m CH CmCHmuCoo CSCHoo HHow CCmm CooHnCm m CH CoHumNHHHnQeaH

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.m mCCme

62

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(ran/Btu) uoueuuaouoo UW

63

evidence for differential uptake between even efficient 652m accumulator
species. In”; no actual mass measurements were recorded. Figure 11
serves as a contrmfl.for Figure 10 to show the uniformity of 65Zn
distribution in the agar plate. Figure 11 is an autoradiographic
exposure of x-ray film by a sterile control plate which represents the
background exposure of x-ray film by the 652n in the agar medium for a
longer film exposure time than that used in Figure 10.

The uniformity of exposure shown in Figure 11 is contrasted with
an apparent zone of 652n depletion from the agar medium near the site of
colony accumulation shown in Figure 10. The zone of 652D depletion from
the agar 1118le is shown very clearly in Figure 12 which represents
reconfirmation of 65‘Zn immobilization by an isolated pure culture of

Penicillium using the autoradiography plate technique. The single large

 

65Zn immobilizing colony represented by the dark area in the center of
autoradiograph shown in Figure 12 was the result of isolating and
culturing a single 652n immobilizing colony similar to one of those
shown on Figure 10. Similar colony isolation and reconfirmation of 65Zn
immobilization was accomplished for a number of individual colonies.

Pure tube cultures were established from the agar plates on which
65Zn immobilization had been reconfirmed.

Once the pure tube cultures were well established, phase contrast
uncrosc0py was combined with standard microbiological staining and
related identification techniques to tentatively identify as many of the
species of microorganisms as possible. Photomicrographs were taken of
representative slide material and the results are species of the

following: Penicillium, (Figure 13), Paecilomyces (Figure 14), Fusarium

 

 

64

(Figure 15), Cladosporium (Figure 16), Aspergillus (Figure 17), Mucor

 

(Figure 18), Rhizopus (Figure 19), Bacillus (Figure 20), and a culture
of an unidentified gram-positive rod-shaped bacteria (Figure 21).

Isolates of Penicillium (Figure 13) were the most abundant even though a

 

number of fungi tended to be represented as 652n immobilizers. The most
abundant 65Zn-immobilizing bacteria developing in these plates were

spore-forming Bacillus £33., and another unidentified gram-positive rod

(Figures 20, 21).

 

65

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66

 

 

 

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.HH meste

68

 

 

 

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.qH muswfim

74

 

 

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80

 

81

 

 

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83

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SUMMARY

A soil perfusion system was used to produce a population of soil
microorganisms that could significantly reduce the level of Zn in soil
solution to a level lower than that attainable by soiltflmmdcal and
physical factors.

Soil was sterilized in a nondestructive manner by using 60cc gamma
radiation. The investigation produced data on soil and microbial
inactivation as a result of time of perfusion and level of microbial
activity where differing levels of microbial activity were obtained by
stimulating microbial populations. Microbial populations were increased
by adding increasing amounts of nutrient broth for energy euui nutrient
sources.

The microbial populations did respond to energy and nutrient
additions and the amount of Zn which could be inactivated by a soil with
a high level of microbial activity was significantly greater than that
which could be inactivated by soil without measurable biological
activity and the additional inactivation was due to microbial
immobilization. The enhanced Zn inactivation by soil generally reached
a new plateau (Zn level in perfusion solution) within 48 to 72 hours
after the addition of an energy and nutrient source to the perfusion
system.

Subsequent tests using radioactive Zn (652n) and autoradiography
along with standard microbial plating techniques demonstrated that only"
certain microorganisms were involved in the elemental uptake. These

could be isolated as colonies in pure culture, plated out again on

89

9O

65Zn-containing nutrient medium and be reconfirmed as Zn immobilizers.
These reconfirmed isolates were tentatively identified as Species of
Penicillium, Paecilomyces, Fusarium, Mucor, Cladosporium, Aspergillus,

Cephalosporium, and Rhizopus. Isolates of Penicillium were the most

 

abundant. The most abundant Zn-immobilizing bacteria developing in
these plates were spore-forming Bacillus _s_p_., and another unidentified

gram-positive rod.

can

4.

CONCLUSIONS

From the results presented in this study the following conclusions

be drawn:

The soil perfusixnm apparatus is a useful tool for static, dynamic
studies and in evaluating the role of environmental variables on
biological and nonbiological processes in the soil.

Addition of nutrient and energy sources for microbial growth and
activity, especially in sandy soil, increases the Zn
immobilization capacity by increasing the activity of the
microorganisms.

Removal or immobilization of Zn by direct microbial uptake appears
to be relatively unimportant in organic soil. Similarly, in
Brookston clay loam the presence of microbes and energy and
nutrients is of minor importance in Zn uptake because soil
chemical and physical factors control the level of Zn in the soil
perfusion solution.

Radioactive Zn and autoradiography along with standard microbial
plating techniques can be used to isolate and confirm colonies of
microorganisms that immobilize Zn.

The colonies of Zn-immobilizers isolated in pure culture were

identified as species of Penicillium (most abundant),

 

spore-forming Bacillus sp., and another unidentified gram-positive

rod.

91

92

Only 21% of the platable microorganisms in the soil tested
(Rubicon sand) were Zn immobilizers.
Variations in the capability of Zn immobilization exist between

different and even the same species of microorganisms.

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