The Physical Environment in Soil Microbiology: An Attempt to Extend Principles of Microbiology to Soil Microorganisms

THE PHYSICAL ENVIRONMENT IN SOIL MICROBIOLOGY: AN ATTEMPT TO EXTEND PRINCIPLES OF

MICROBIOLOGY TO SOIL MICROORGANISMS

Authors:

Referee:

Tsutomu Hattori Reiko Hattori Institute for Agricultural Research Tohoku University Sendai, Japan

A. D. McLaren College of Natural Krsources University of California Berkeley, California

I. INTRODUCTION

ln spite of the exciting development of microbiology in the last 100 years, there may be little doubt that our understanding of microbial life in complex natural environments, particularly in soil, is far from being based on principles of this art of science. Microbiological studies have been carried out mainly with pure culture under well-defined conditions, and many microbiologists dislike complex natural environments. However, natural environments such as soil offer great challenge to microbiologists inquiring into dynamic features of the microbial world in relation to its environment and the way to control it.

In contrast with pure cultures, in which attention has been focused mainly on the chemical composition of their media, the physical constitution of the environment wiU have prominent importance in the case of soil microorganisms. Soil is a heterogeneous, discontinuous, and structured environment dominated by a solid phase varying in size from less than 0.2 pm to greater than 2 mm. This discontinuity and variability in particle size may result in soil being a composite of enumerable

small microbial communities, each circumscribed by its own immediate environment, as suggested by Stotzky.'

To facilitate description of any part of the complex environment in relation to microbial life, i t is attempted in this presentation to introduce a number of concepts on physical constitution of soil. Thus, various levels of physical concepts (such as solid-liquid or liquid-air surface, colloidal particles, soil aggregate and soil profiles) may be discussed as basic environmental components of soil microorganisms. Through stepwise recons truc- tion of the physical environment, the authors will try to extend microbiological principles to soil microbiology (Figure 1). This aspect is a part of our system of analysis of soil microorganisms.'

11. SOLID SURFACE

Microbiology has developed mainly on the basic assumption that each cell is surrounded by a three-dimensionally continuous and homogeneous environment; that is, nutrient solution or other homogeneous systems. However, most microbial cells are believed to be placed at the solid-liquid

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FIGURE 1. Stepw ise reconstruction ot' the physical environment of soil microorganisms. Microorganisms in liquid medium ( I ) may be affected by a solid surface (2) , and then colloidal clay particles (3) . Whcn soil colloid particles are aggregated, microorganisms may be distributed in a great number of discrete microhabitats (4). The life af soil microorganisms in siru may be controlled by flow of water and pas through soil profiles (5).

Consequently, the first task of this investigation is to determine whether (and in what manner) the physiological behavior of microbial cells would change if they were transferred from bulk solution to the interface. Considering the scope of the subject matter, only major topics of importance for the extension of microbial principles to soil microorganisms will be considered. The reader's attention is directed to excellent review articles by Marshall6 and Zvyagintsev7 regarding this problem.

A. Surface Area in the Soil Compared with the physical environment of a

pure culture system, one of the surprising and important characteristics of soil is the large amount of surface area per unit mass. Soil is essentially a dispersed system; that is, a system in

which the particles are in a fine state of subdivision or dispersion. The specific surface of a dispersed system is taken as the sum of surfaces of the constituent dispersed particles, referred to as unit volume or weight of the substance. The specific surface is a geometrical concept and largely depends on the shape and size of the constituent particles. Figure 2 shows that a very small amount of substance may have an extra- ordinarily large surface area if a sufficiently high degree of subdivision is realized. The same volume of liquid medium in a test tube is circumscribed by only several square centimeters of glass surface.

B. Observations on Effects of Solid Surfaces on Microbial Life

Surface phenomena are complicated and difficult to analyze in the context of complex solu-

424 CRC Critical Reviews in Microbiology

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-3 -4 -5 0 -1 - 7

Dianeter of par t ic le i cm, logarithms )

t."CURE 2. and particle size.

Relation between the specific surrace area

tions used in cultivated microorganisms or those found in soil. However, gross effects of such interactions have been observed in many systems.

I . Soil Microbiologists' Observations Since the early period of the history of soil

microbiology, researchers have paid attention to surface. They showed that by adding various substances (such as clay, sand, charcoal, brick dust, bot tom mud, or glass) into culture media, these solid surfaces profoundly affected microbial activity and growth. Some of them were depressive and others stimulative. Depressive effects included depression of biological activities by fine solid particles,' depression of COz evolution from the cultures of several kinds of Bacillw by addition of soil particles,' and decrease in COz evolution by Azutobacter clzruucocciiini ' or other bacteria.' ' Evidences for stimulative effects of solid surfaces were also reported on nitrogen fixation by Azoto- bacter with addition of soil and on petroleum, alcohol, and urea oxidation by the addition of charcoal and silica.' Stimulation of sulfate reduction by adsorption' and enhancement of growth of several soil bacteria in the presence of clay mherals or other colloidal substance^'^^' were also reported.

These apparently contradictory observations as t o whether a solid surface is stimulative or harmful to attached bacteria cannot strictly be discussed

witliout considt'ratiw1 U I the nature ot the surtace or other experimental conditions. The tendency generally has been that particles larger than bacteria are more beneficial than those that are smaller. The latter case was attributed t o the reduction of the effective surface area of a bacterial cell for nutrient absorption or respiration, while beneficial effects of solid surfaces of larger particles was attributed to the concentration of nutrients and the removal of toxic substances around attached bacteria.

More recently, Lees and Quastel, introducing the soil percolation technique t o soil microbiology, made kinetic studies of nitrification in soil. They showed that the rate of ammonium ion oxidation of a given quantity of ammonium sulfate was a function of the degree t o which ammonium ions were adsorbed on or combined in the soil in the form of the soil's base exchange complexes; the greater amount of adsorption, the faster the oxidation. These results were interpreted to mean that nitrifying bacteria grew on the surface of the soil crumbs at the site where ammonium ions were held in base exchange combination and proliferated a t the expense of such adsorbed ammonium ions. Although the concept may be only applicable to ammonium- oxidizing bacteria,' ' ' the work again stimulated research interest as t o relationships between solid surface and microbial life.'

2. Marine Micro biologists' Observations Extensive studies concerning the surface effects

were also carried out by marine microbiologists. ZoBell investigated the influence of the volume of sea water in glass containers of similar shapes on the multiplication of bacteria in stored sea water and noted that bacteria in smaller containers grew more intensively.' ' The result suggested stimulative effects of solid surface on bacterial growth, especially in the case of sea water and other raw waters which naturally contain only very low concentration of nutrieI.ts. Taking his own and others' results into consideration, he summarized characteristics of adsorptive phenomena between solid surface and bacteria in sea water or other natural waters as foIlows.' '

1. A beneficial effect of solid surface is usually evident only in very diluted nutrient solution (less than 10 mgil).

2. Such a beneficial effect seems t o be

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related to the accumulation of nutrient on the surface by adsorption and also to retardation of the diffusion of exoenzymes or hydrolysates away from the cell which promotes assimilation of nutrients.

3. Many bacteria found in sea water are sessile, growing exclusively or preferentially attached to a solid surface. Most of these bacteria appear to grow on a solid surface by exudating a mucilaginous hold fast and a few have stalks.

The evidence for the beneficial effect was at first clearly shown in the experiment of Heukelekian and Heller'' who studied the effect of glass beads on the growth of Escherichia coli in relation to nutrient concentration. They showed that the limiting concentration of nutrients for the growth of the organism was lower in the case of the experiment with glass beads than that without them.

As to the third point mentioned by ZoBell, i t may be important to notice the life of Chufobacter. Their wide occurrence in seawater, other natural waters, and even soil has been recognized. These organisms secrete holdfast material at the flagellated or stalked end and easily attach to glass, collodion, or cotton fibers, as well as one another (leading to the formation of rosette) or other organisms.' ' In addition to Caulobacter, there are many observations that in natural water various kinds of bacteria grew tenaciously attached to a solid surface.'

3. Evidences from Continuous Flow Culture Experiments

Recently, evidences on sorption phenomena of bacteria were presented from physiological studies of pure cultures in continuous flow system. I t has been noted that the data from continuous flow culture experiments deviate apparently at a higher flow rate; bacterial concentration was higher and substrate concentration was lower than the values calculated. It was suggested that the existence of steady states, at dilution rates greater than the critical rate, was attributable primarily to incom- plete mixing or medium inflow and to apparatus effects such as detachment of growth from the glass wall.'

Larsen and Dimmick suggested by their experiment that the growth on the walls of a culture vessel can be a highly significant factor in the interpretation of continuous flow culture

kinetic^.'^ The results of their experiment are as follows.

1. When the dilution rate was temporarily increased to 7 or 32 vol/hr, the average cell density was extremely reduced. However, after flow was stopped, only a few minutes more were required to return to the original level. When a portion of a culture was transferred at the time of stop to an identical vessel, such an abrupt rise in cell density was not observed.

2 . The cell density in the effluent changed with dilution rate, but the total output of cells per hour remained constant. They also suggested that there is a build-up of sorbed cells until the surface density reaches a maximum value which is characteristic of both the bacteria and the surface involved. The value was lo6 cells/cm' with E. cofi and glass and lo8 cells/cm2 with Serratia marcescens and glass.

Topiwala and I-lamer formulated Larsen and Dimmick's suggestion based on the following assumptions.'

1. Bacteria adhere to the walls of the vessel in contact with the culture and form a monolayer.

2 . The total mass of bacteria sorbed to the walls remains constant after an initial build-up period. Thus the progeny of the sorbed bacteria are released into the culture.

3. The growth rate of attached bacteria is the same as that in the suspended culture.

Thus, they obtained the following relations between biomass (X) and limiting substrate concentration (S) in a steady state:

where

p = the specific growth rate; y = the yield factor; S, = the incoming substrate concentration; D = the dilution rate; K = the constant value determined by the

total mass of sorbed cells.

As to the growth pattern of attached cells on the surface, Helmstetter and his colleagues


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proposed that if a population of bacteria could be irreversibly bound to a surface while growth medium flowed past the surface, the only cells which would appear in the medium would be those new daughter cells which were not involved in the attachment of their parent cells to the surface. Since the cells bound to the surface would be growing, the bound daughter cells which eluted from the surface would be the youngest cells in a lag phase culture.2 ’-* Applying this proposition, they succeeded in obtaining synchronized populations.

C. Molecular Environment a t a Solid Surface Physical states of molecules and ions in a

solution are greatly influenced by a solid surface and, consequently, the molecular environment of microorganisms at the surface may be different, more or less, from that in the bulk solution.’

i. Adsorption Phenomena An important property of surfaces is adsorp-

tion: the existence of a higher concentration of any particular substance at the surface of a liquid or solid than is present in the bulk. A significant equation describing adsorption at the surface of a solution is Gibb’s adsorption equation. For a dilute solution of concentration (C) the equation may be written as:

(3 )

where

S = the excess concentration of the solute per square centimeter of surface, as compared with that in the bulk solution;

dy/dc = the ratio of increase of the surface tension of the solution with the concentration of the solute;

R = the gas constant; T = the absolute temperature.

According to the equation, any solute which causes the surface tension of the solvent to decrease (that is, dr/dC is negative) will have a higher concentration in the surface than in the bulk solution, since S will be positive. On the other hand, if drldC is positive (the desolved substance raising the surface tension), the S will

have negative value. The concentration of the solute will thus be lower in the surface than in the bulk. The latter case is known as negative adsorption. In consequence of adsorption phenomena, the molecular or ionic environment of microbial cells at a surface may be quite different from t h d in the bulk solution, which may (more or less) result in changes of physiological activities of the microbial cells.

2. Surface Potential The molecular mechanisms of adsorption are

not yet well known, but may involve complicated factors. The surface potential $ may be one of the important factors. The distribution of an ion in a solution is governed by a Boltzmann relation: at places of positive potential, anions are concentrated and cations are repelled, whereas for places of negative potential the reverse is the case.

(Ci), = (Ci)b . exp(-zjC*/kT) (4)

where

Ci = the concentration of an ion of kind i at a point where the potential is $;

s = the concentration at surface; b = the concentration in the bulk; zi = the valence of the ion; e = the charge of proton; k = the Boltzmann constant.

Thus, ions acting as microbial substrates or inhibitors may be condensed or diluted near a surface which will affect the microbial or enzymic activity-substrate or activity-inhibitor concentration relationship: insertion of Equation 4 into the Michaelis-Menten equation gives;

where

v = the rate of the enzyme reaction in the bulk;

vs = the rate of the enzyme reaction at the surface;

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vmaX = the maximum rate; k, = the Michaelis constant; S = the substrate concentration.

The apparent Michaelis constant of the enzyme reaction at the surface (Km,s) can be expressed by :

3. Surface p H Microbial growth and activity are largely

influenced by hydrogen ion concentration. Hartley and Roe obtained an equation defining the local concentration of hydrogen ion near a surface (H+)s.3 According to the Debye-Huckel theory, the surface potential, +, can be replaced by the electrokinetic potential (zeta potential, {) and, thus, Equation 4 is rewritten as:

McLaren and his colleagues showed that the optimum pH for the activity of enzyme adsorbed on negatively charged surfaces was observed a t higher pH value as compared with that of the enzyme in the bulk solution.’ ’ McLaren and Skujins also noticed that nitrification in soil took place at a hydrogen concentration as high as pH 4, although in solutions there is no nitrification below pH 6 . They interpreted the difference by the pH s h f t at soil surface.’

4. Surface Eh Many microbial reactions involve oxidation-

reduction processes which are closely connected with the redox potential (Eh). The potential is given by :

RT (A ) ’ / * (H*) I h = E * + - In F AH,)^/^

where

(10)

Eo = the standard redox potential: F = the Faraday constant: R = the gasconstant; A = t he c o n c e n t r a t i o n of oxidized

molecule or ion:

AH, = the concentration of reduced molecule or ion.

As mentioned above, the concentration of H’ ion is greatly influenced by the surface potential; if AH2 and A are ionic forms, their concentrations are also affected by the potential. Consequently, the Eh value at the surface may also be different from that in the bulk solution.’0s

D. Relationships between Microbial Cells and Solid Surfaces

The whole problem in relationships between microbial cells and solid surfaces may involve a variety of microbial behaviors and their interactions with surfaces: Brownian movement, flagella movement, tactic movement, hydrophobic effect, physical interactions between cells and surfaces, cell adhesion and detachment from surfaces, etc.

1. Brownian Movement A suspended colloidal particle such as a micro-

bial cell is considered to be in kinetic equilibrium with the molecules of medium. Thermal movements of the latter result in the Brownian movement of the former particle. The basic idea gives the following equation:

(1 1) n ’r (1/2 MV’) = (1 /2 mV‘) = 3/2 -N

where

M = the mass of colloid particle; m = the mass of molecule of the medium;

= the mean of the square of velocity of colloid particle;

v = the mean of the square of velocity of molecule of the medium;

N = Avogadronumber.

-

Kinetic energy of each freely moving element is therefore completely determined by temperature. Several authors observed that nonmotile cells were sorbed on glass surface through Brownian move- rne r~ t .~” ’

2. Flagella Movement Motile organisms are distributed in most of the

phyla of the microbial world. Many motile microorganisms can move actively in liquid medium by means of locomotive organelles known as flagella


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or cilia. According to Marshall et al.,33 most motile bacteria frequently exhibited a peculiar rotational behavior at a liquid-glass interface. Such bacteria sorbed at the pole of the cell (an edge to surface association) and, by virtue of the motive force of the flagellum, rotated violently around the fixed pole. If sorbed in a face-to-face position, they rotated in either direction in a propellerlike fashion.

3. Tactic Movement A tactic response means the movement of an

organism toward or away from a source of stimulation. Many studies have been done on the chemotactic response of bacteria to oxygen, inorganic salts, and a great number of organic compounds including organic acids, amino acids, carbohydrates, peptone, meat extract, and proteins. Often a substance caused a positive response (movement toward the source of stimulation) when applied at a small concentration, whereas a negative response occurred when the substance was more concentrated at a surface. Such chemotactic movement might be involved in microbial sorption on a surface.

4. Hydrophobic Effects Marshall and C r ~ i c k s h a n k ~ ~ observed that

individual cells of Flexibactrr aurantiaciis and Hyphomicrobium vulgare orientated themselves perpendicularly to the interface in air-water, oil- water, and solid-water systems. They suggested that the orientation may result from a relatively hydrophobic portion of each cell being rejected from the aqueous phase of the system.

5. Interaction between Microbial Cells and Surfaces

The energies of interactions between a microbial cell and a surface may be expressed by a rearranged version of the formula derived by Hog et a1.3

VT = VR + VA (12)

where

VT = the totalenergy; v, = the repulsive energy; v, = the attractive energy, as defined by:

where

a = the radius of curvature of the particle and suffixes refer to each particle;

J / , and G 2 = the surface potential of each particle;

K = the inverse Debye-Huckel length; H = the distance of olosest approach

of the two particles; E = the dielectric constant; A = Hamaker's constant.

The series of curves in Figure 3 shows the increase in magnitude of the resultant repulsion curves and the increase in particle separation with decreasing electrolyte concentration. The pro- gressive increase in repulsion energy with decreasing electrolyte concentration closely parallels the decrease in reversible sorption of a bacterium to the surface. Such decrease was observed in sorption of Achromobacter to glass surface by Marshall et al.33

At high electrolyte concentrations a secondary minimum (attraction trough) is apparent. As to this effect, it is of interest that Zvyagintsev described an accumulation of bacterial cells near surfaces.

6. Sorption of Microbial Cells on a Surface Marshall et al.33 recognized two distinct phases

of bacterial sorption on a glass surface: the instantaneous reversible phase and the time- dependent irreversible phase. They noticed that reversible sorption of a nonmotile Achromobacter strain decreased to zero as the electrolyte concentration decreased. The reversible phase was interpreted in terms of the balance between the electric double-layer repulsion energies at different electrolyte concentrations and the van der Waals attraction energies, On the other hand, the irreversible phase of sorption implies a firmer adhesion of bacteria to a surface. Polymeric bridging between the bacterial ce11 and the surface of the tested

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: Part i c l e se p a ra c ion

FIGURE 3. Interaction energy as a function of particle separation. Electrolyte concentration is higher with curve 1, mediated with curve 2, and lower with curve 3.

material might overcome the repulsion barrier between these two surfaces. In observations by Marshall et al.33 it is interesting to note that extremely low levels of available carbon stimulated irreversible sorption, while higher levels inhibited this process.

The physiological age of cells also largely affects their ~ o r p t i o n . ~ 93 Zvyagintsev described three types of influence, as shown in Figure 4.’

As the possible forces of sorption between cells and surfaces, the following list by P e t h i ~ a ~ ~ is available.

1, Chemical bonds between the opposed sur-

2 . Ion-pair and ion-triplet formation. 3. Forces due to charge fluctuations. 4. Charge mosaics on surfaces of like or

5 . Charge attraction of opposite signs. 6. Electrostatic attractions between surfaces

7. Electrostatic attraction due to image

faces.

opposite overall charge.

of like charge.

forces.

. .

0 5 10 15 20 25 30 35 40

Time ( hours )

I-’IGURE 4. Effects of culture age on bacterial cell sorption on the glass surface. Solid lines indicate growth of bacteria and dot ted lines adsorbability of cells at each growth phase. (From Zvyagintsev, D., Interaction between Microorganisms a r d Solid Surfaces, Moscow Uni- versity Press, MOSCOW, 1973.)

8. Surface tension or surface energy. 9. Van der Waal‘s forces.

10. Charge repulsion between surfaces of like

1 1. Van der Waal’s forces of repulsion. 12. Hindrance to attraction due to steric

barriers such as inert capsules and solvated layer.

charge.

There are various modes of cell sorption to surfaces, as shown in Figure 5.7

Sorption of microbial cells is very selective. ZoBell showed that among 96 isolates from sea water, 29 strains adhered to glass surface markedly, 47 strains did not at all, and 20 strains variably. He did not find any relationship between the Gram reaction of these strains and their


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FIGURE 5 . Various types of bacterial sorption on solid surfaces. (From Zvyagintsev, D. , Interaction between kficroargariisnis and Solid Surfaces, Moscow University Press, Moscow, 1973.)

attachment properties.' Larsen and Dimmick also noted the selectivity of bacterial sorption to the walls of a glass vesseLZ3 Zvyagintsev summarized his extensive experiment on selective sorption of bacteria to glass surface (Table l ) . 7

Marshall et al. noticed that the phase of firm or irreversible sorption was a highly selective process. Many of the bacteria initially attracted to the surface (reversible sorption) did not become firmly adhered to the surface (irreversible ~ o r p t i o n ) . ~

7. Detachment of CeIl from Surface Sorption and detachment are not quantitatively

reversible. For example, interfacial properties of cells (such as their electrophoretic mobility), while directly influencing cell contact and probably sorption, will not always directly influence cell d e t a c h m e n t by electrostatic repulsion, If irreversibly sorbed cells detach from the surface, the plane of separation will run through the material having the lowest cohesive strength. Sometimes pieces of cell remain stuck to the surface; sometimes fragments of the solid adhere to the cell, as pointed out by we is^.^

E. Microbial Activity and Growth on a Surfme I . Possible Factors Affecting the Activity of Cells on a Surface

Although the literature contains apparently conflicting reports of both stimulatory and inhibitory effects of solid surface on microbial activity, the following factors may be involved in these effects.

1 . Concentration or dilution of substrate at the surface.

2 . Change in the value of pH or Eh a t the surface.

3. increase or decrease in inhibitor concentration at the surface.

4. Masking of cell surface by solid surface.4

5 . Change in the apparent activation energy? '

6. Release of metabolically active molecules or ions from the cells.4 I ,4

2. General Aspect of Microbial Growth on a Surface

In the study of microbial growth on a surface,

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TABLE 1

Adsorption of Various Bacteria on the Surface of Glass

model, presented by Helmstetter et al., is that a cell may be sorbed to the surface at its pole, and after elongation towards the bulk solution, only newly divided daughter cells detach from the

adsorbed adsorbed* surface’ (Figure 6A). The second model, presented bv Larsen and DimmickZ3 and Touiwala and

Strains Strains strongly

Number of strains

Hamer24 and developed by H a t t ~ r i ? ~ 9 4 5 is that bacterial cells sorbed to the surface may multiply

Genusofbacteria tested No. ”/O No. % on the surface, and newly divided daughter cells may remain in the sorbed state until bacterial Actinomyces 20 14 70 1 5

Mycobacterium 10 6 60 4 40 density on the surface reaches a maximum value Micrococcus 18 13 72 7 39 which is characteristic of both the bacteria and the Sarcina 12 1 1 92 8 67 surface involved. After having reached to the

I

Bacillus 43 27 63 maximum value, daughter cells may be released into the culture medium. Larsen and Dimmick Pseudo mo ms 26

Rhizobium 8 suggested that, in the case of Serratia marcescens

23 89 16 62 12

*“Strains strongly adsorbed” are those sorbed more than lo6 cells/cm2.

and glass surface, the maximum value may be 10’ cells/cm2. In the case of E. coli and the surface of

F r o m Zvyagintsev, D., Interaction between Microorganisms and Solid Surfaces. Moscow University Press, Moscow, 1973.

it is essentially important t o take into consideration that a surface is two dimensional, contrasting with a three-dimensional liquid medium. In addition to the factors affecting enzymic and bacterial activity on the surface, the following points should be considered.

1. The discrimination of a free and a sorbed state of microbial cells and their reciprocal transi- tion.

2. The pattern of arrangement of cells on the surface.

3. Growth constant specific to the cells in the sorbed state.

ion exchange resin Dowex I (in chloride form): ’ the value will be discussed in Section II.E.5. The second model is depicted in Figure 6B.

Another type of cell detachment from the surface was also ~bserved .~’ Detachment of cells may also be caused by some autolytic process or other drastic physiological change, which is observed in a later stationary period in batch culture but not in continuous culture.

3. Kinetics o f Bacterial Growth on a Surface This section is concerned with the general

kinetic principles relating to the growth of bacteria sorbed to a surface. In particular, equations will be developed on the growth in continuous flow culture in which cells detach as in the second model.

To describe microbial growth in a continuous culture containing dispersed solid particles, two basic equations should be written. Equation I defines the growth on the surface and Equation I1 defines bacterial in liquid medium continuously flowing.47

Equation I = increase of cells on the surface = (increase due to growth on the surface) (increase due to sorption from medium).

As to the first point* we define Only irreversibly sorbed cells in the sense of the terminology of Marshall et al.33 as those in the sorbed state. The second point concerns two questions: whether the cells sorbed to the surface at their pole or face and whether the Cells adsorbed on the solid surface as a monolayer or m~l t i l aye r .~ dX

It was established that when cells in the sorbed Gs = (1 - f ) ~ s X s + K , ( l - f ? X b

state were incubated in a nutrient medium both as a batch culture and a continuous flow culture, a great amount of cells were detached from the s ~ r f a c e . ’ ~ 3 4 4 As to the mechanism of the detachment of cells accompanied by cell growth on the surface, two models were presented. The first (decrease due to flow out).

Equation 11 = increase of cells in liquid medium = (increase due to growth in the liquid) + (increase due to detachment of newly divided daughter cells) - (decrease due to sorption to the surface) -


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FIGURE 6. Growth patterns of bacteria on a surface. (A) Helmstetter's model.' (B) Cell elongation along the surface, which is, if the adjacent area has been occupied by other cells, followed by detachment of the newly divided cell. (C) Spore (striped sphere) detachment due to expulsion by the adjacent elongating cell. (D) Spore formation in a sorbed state.

I dA = ! J b X b + f p s X s " . - K,(1 - O X b - D X b (16) dt

where

Xb =

xs =

- pb -

- I-1, -

Ka = f =

D = v =

cell numbers per unit volume of the liquid medium; cell numbers per unit area of the surface; specific growth rate of the cells in the bulk solution; specific growth rate of the cells on the surface; a constant relating cell sorption; a function defining detachment of cells from the surface; dilution rate of the continuous system; volume of the liquid medium in the culture1 vessel.

We may expect that if cells sorbed on the surface in a monolayer, f would be a function of Xs/X,,max, where X,,,,, is the maximum value of cell density in the monolayer; the value of f would be very small (nearly 0) until Xs/Xs,max is less than 1. When Xs/$,max approaches 1, the value of f would also increase rapidly to 1.

In a steady state, Equations 15 and 16 are

From Equations 17 and 18 we can obtain:

When the surface is saturated with cells, i.e.,

Other growth constants of sorbed cells, such as growth yield and the saturarion constant, would also be different from those suspended in liquid medium.

4. Kinetics of Spore Formation of Sorbed Cells in a Continuous Culture System

Dawes and Thornley presented a formulation of bacterial spvrulation in a continuous c u l t ~ r e . ~ ' They assumed that a sporulating bacterial population consists of three categories; (1) vegetative cells, A,which have not been initiated to form spores and 3re undergoing the normal process of cell division; ( 2 ) population B, which consists of cells that have become initiated to form spores, but which have not matured; and (3) population C, consisting of spores which have matured. Population A can undergo one or two competing processes: growth with the specific growth rate, P, or sporulation with specific sporulation rate, 77 (the rate of the reaction A + B).

Based on the Dawes and Thornley equation, Hattori attempted to formulate spore formation of bacterial cells on a s~rface.''~ She assumed further that:

1. The surface may be covered with vegetative cells and spores in monolayer, and the maximum number, N,,,, may be determined by the sum of the number of vegetative cells (X,) and that of spores (S,) on the surface.

2. The rate of spore formation on the surface may be different from that in the bulk mediuni.

3. There may be two cases in cell detachment due to the multiplication of spore-forming bacteria in the monolayer with the maximum cell

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number N,,,. When the cell elongates towards another vegetative cell placed at the nearest neighbor, the newly divided cell may be released into the liquid medium; when the cell elongates toward spores sorbed at the nearest place, the spore may be replaced by the newly divided cell and released owing to weaker sorption (Figure 6C).

4. A spore formed from a vegetative cell on the surface may remain in the sorbed state until being replaced by a newly divided cell.

5. The rate of germination may be neglect- ed.

In a steady state the number of vegetative cells (X,) and that of spores (S,) on the surface may be given by :

= (newly divided cells substituted for spores) - (spores newly formed from vegetative cells)

d Xs - d t

= (newly formed spores) - (spores detached due to the replacement by newly divided cells)

d S s - d t

= vs x, - f i s ox,

where

1.1, = the rate of growth of vegetative cells on the surface;

us = the rate of spore formation of vegetative cells on the surface;

P = a function determining the probability at which a spore would be replaced by a newly divided cell.

The function may, in a steady state, be given from Equation 22 or 23

The relation was supported by the observation that the ratio of the number of spores to that of vegetative cells on the surface remained almost

constant during cultivation of Bacillus subtilis sorbed on an ion exchange resin, Dowex 1, in a continuous flow system?

Kinetics of spore formation as a whole in a continuous flow system containing sorbed cells or spores can be established on the basis of the Dawes and Thornley equation and Equations 22 and 23.4

5. Physiological Characteristics of Bacterial Cells Sorbed on an Anion Exchange Resin, Dowex 1

The results of our study on chemical activities may of bacterial cells sorbed on the resin3 ‘ 34 O4

briefly be summarized.

1. Rates of oxidation of various substances were depressed by cell sorption to 1 to 30% of free cells and recovered to 40 to 70% by desorption of cells from the surface.

2. Activity-pH curves shifted to the alkaline side by about one unit by cell sorption and returned almost to the original curves (with free cells) by cell desorption.

3. Activity-substrate concentration by curves shifted to ten times higher concentration cell sorption in the case of anionic substrate and did not in the case of nonionic substrate (such as glucose) and amphoteric ion substrate (such as alanine or asparagine).

4. Duration of the lag time observed in the case of induced oxidation of organic substances was markedly diminished by cell sorption, and the effect remained still with desorbed cells. Heat extract from free cells greatly prolonged the lag time of desorbed cells.

5. Activity-temperature relationships also varied more or less by cell sorption.

To interpret statements 2 and 3, the authors tentatively presented a concept of cationic layer circumscribing sorbed cells, although the results might be interpreted in terms of other electric effects.

As to growth of bacteria sorbed on the resin, it was noticed that when sorbed cells were incubated in a continuous flow system (D = 1);’ cell concentration in the effluent at first increased stepwise with periodicity (first phase) and cells in the effluent showed a synchronous growth when they were transferred into a new medium in a closed system (Figure 7). As a result of further incubation of the continuous system, cell concentration of the effluent came to keep a constant


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0

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1 2 3 4 5 6 7

Time ( hours )

F I G U R E 7. (A ) Increase of cell concentration in flowing liquid medium when E. coli cells sorbed on an anion eschanger, Oowes 1 (in C1 form) were incubated in the flowing medium (D = 1). An arrow shows that a part of effluent was reincubated in batch system. ( U ) Growth of the effluent in a batch culture. (From Hattori, R., J . G ‘ c x Appl . hficroDioI., 18, 319, 1972. With permission.)

level (second phase). I t was considered that the periodicity in the first phase may reflect the age distribution of sorbed cells, and the period may correspond to the mean generation time of the population. The specific growth rate on the surface was obtained from data in the second phase and compared with that in the bulk solution (Table 2 ) .

I t was noted that bacterial density of the surface was kept constant in a steady state of the continuous culture, although the density in a batch culture varied widely during incubation. The variation of cell density of the resin surface in a batch culture may be attributable to changes of adsorbability resulting from modification of cell surface structure or stimulation of autolytic enzyme in sorbed cells. The cell density was estimated to be ca. 2.3 X lo7 cells/cm* in a continuous culture and 8.8 X lo7 cells/cmZ in a batch culture (the latter is a maximum value) by electron microscopic observation by carbon replica method.44745 The existence of a maximum number of growing cells on the surface was also supposed by Figure 8. The apparent lag time of

cells growing on the surface decreased, in inverse proportion to initial cell density, to a value which would be the “true” lag time or the lag time of cells at the maximum cell density on the surface.

A steady state of continuous culture of spore- forming bacteria, B. subtilis, was also realized. The rates of growth and spore formation with sorbed cells were compared with those in the bulk nutrient solution (Table 2).49

I t is interesting that although oxidations of various substances were depressed by cell sorption, growth rate and spore formation rate were markedly accelerated by cell sorption to the surface. However, it should be mentioned that these effects would vary with both the type of bacteria and surface.

111. COLLOIDAL PARTICLES

Soil is essentially a colloid dispersed system. The colloid-size particles play an important role in determining the physical or physicochemical properties of microbial environment in soil. The most important characteristics of a colloidal system can

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0 1 2 3 4 5 6 7 8

Time ( hours )

- 7 . 5 &I

0 s v

a PI U m 5.0 U

In &I .* IL1

a r U

ly 2.5

c '4 U In k

0 a

0 0 0 .5 1l 1.0

x 10 i g - r e s i n I n i t i a l d e n s i t y of sorbed cells

FIGUlZE 8. Relation between initial density of sorbed cells off:. coli and dumtion of the tirst growth step, in which ncwly divided cells sorbed preferentially on the surface and did not appear in the liquid medium (A). (B) Duration of the first step was plotted against initial cell density. (I:roni Hattori, R., J. Gen. AppL Microbid., 18, 319, 1972. With permission.)

TABLE 2

Rates of Growth and Spore Formation in Liquid Medium and on the Surface of an Anion Exchange Resin Dowex I , in the Chloride Form.

On the In liquid media surface

Organism pb Vb f i s us

E. coli 0.54 2.4 B. subtilis 0.41 0.026 0.67 0.52

Note: Dilution rate is 1. I.( and u are growth rate ( h r - ' ) and rate of spore formation (hr-' ), respectively. Suffixes b and s refer to those in liquid medium and on the surface, respectively.

Based o n Hattori." "'

be ascribed to the fact that the ratio of surface area to volume of the particle is extremely large. Consequently, the second task of our attempt is to investigate interactions between microbial cells

and colloidal particles, and microbial behaviors in a colloid system.

A. Colloid Particles as the Basic Physical Constituent of Soil

Soil particles are divided into various size fractions as shown in Figure 9B. The relative proportions of each of these size fractions in any one soil are defined as the soil texture. Figure 9A indicates simplified texture classes.

Soil colloid particles consist chiefly of clay minerals which are reactive. Clay minerals are constituted of distinctly crystalline minerals which are composed of units of alumina and silica. Most clay minerals in soil are platelike in character. Their electron micrographs show that kaolinite has sharp and well-defined edges and montmorillonite has structures ranging from an amorphous- appearing material to extremely thin plates. The clay lattice carries a net negative charge as a result of isomorphous substitutions of certain electro- positive ions by such cations of lower valence. Consequently, the flat layer (face) surface of a clay particle has a constant negative charge. The atomic structure of the edge surface is quite different from that of the tlat layer surfaces; at the edge of the plate, silica sheet and alumina sheet are


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0 b sand

clay

1 c loam

-in 0 / U

1

0 f- sand % -

stones grave 1 s i l t clay

20.0 2 .o 0 . 2 0.02 0.002 mm

FIGURE 9. particles according to their size

(A ) Simplified triangular diagram of soil texture classes. (8) Classification of soil

disrupted and primary bonds are broken. Such a surface carries positive charge in acid solution, due to A13' ions, and a negative charge in alkaline solution with hydroxyl ions, Although silica edge normally carries a negative charge, it becomes positively charged in the presence of very small amounts of A13+ ions. Hence, under an appropriate condition, the entire edge surface area may carry a positive charge.

B. Some Physicochemical Characteristics of Colloidal Clays and Microbial Activities 1. Adsorption and Exchange of Cation by Clays

Colloidal particles of clay are usually charged negatively as a whole. To balance the negative charge, the particles tend to adsorb counterions (cations), which are governed by many factors. The most important factors are type of cations, ion concentration, nature of the anion associated with the cation, and type of the clay particle. As to type of clay, montmorillonite has the highest cation exchange capacity and kaolinite the lowest.

According to Stotzky and his c o l l e a g ~ e s , ~ ~ 35 I

respiration of various bacteria can be stimulated by montmorillonite and also (although to a lesser extent) by kaolinite, which may be primarily the result of clays maintaining the pH of the environ-

ment a t levels suitable for microbial life. The stimulation of bacterial respiration was related to the cation exchange capacity, possibly the specific surface, but not to the particle size of the clays. Since clays apparently exchange H' produced during the metabolism with basic cations for their exchange complex, clays with a high cation exchange capacity are capable of exchanging more H', therefore maintaining longer the pH of the bulk solution at a level suitable for bacterial metabolism.

2. Viscosity and Oxygen Diffusion The viscosity of colloid systems is determined

by three sets of interactions: those between suspended particles, those be tween suspended particles and dispersion medium, and those between molecules of the dispersion medium itself. The sensitivity by whch these interactions can be detected increases rapidly with concentration of the colloidal phase. With respect to their viscosity, colloids are divided into two distinct groups: hydrophile and hydrophobe. The former is characterized by a relatively high viscosity, and the latter does not possess a viscosity appreciably different from that of the dispersion medium. Clay colloid occupies an intermediary position: it is

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hydrophilic because of its hydration and at the same time hydrophobic because of its high sensitivity to electrolyte.

Diffusion of an oxygen molecule and a substrate molecule in colloidal solution is expected to vary in inverse proportion to viscosity of the system, which would affect the activity of microorganisms in it. Stotzky and RemS2 observed that the respiration of a mycelial homogenate of various fungi was generally not affected by montmorillonite or kaolinite at concentrations below 4%, but was markedly inhibited by the fomer clay at a concentration of 4% and above. Kaolinite showed comparable inhibition of respiration only at a concentration above 40%. They interpreted this inhibition of respiration as perhaps related to the viscosity of the medium which, in turn, probably influences the rate of oxygen diffusion.

3. Adsorption of Organic Substances by Clays Organic anions may be adsorbed at the edge of

the clay particles; organic cations, on the contrary, may be adsorbed on the negative face surface of the clay, which is supported by the much larger adsorption capacity of the clay for these cations and by the increase of the basal spacing of montmorillonite clays after treatment with organic cations. Many organic compounds with a dipole character are adsorbed on the layer surfaces and probably also on the edge surfaces of a clay particle.

According to McLaren,’ proteolytic enzymes may be adsorbed on the clay-protein complexes and hydrolyze adsorbed proteins. An entirely new form of the chemical kinetic equation is required t o handle enzyme reactions on insoluble substrates. Upon adsorption of protein, the clay expands as the protein enters the interlayer space of the crystal lattices. As well as the protein adsorbed on the outside surfaces of clay particles, the protein present in the interlayer space is also utilized by microorganisms, suggesting that extra- cellular proteolytic enzymes have access to the interlayer space.

The hydrolysis of clay-protein complexes by enzyme proceeds generally somewhat more slowly than the reaction in solution. A very sharp reduction is caused by drying. Ligno-protein complexes are also highly resistant to hydrolysis. Stotzky’ showed that chymotrypsin and lactoglobulin were utilized by bacteria when complexed with montmorillonite, but to a

considerably lesser extent than when not complexed. Catalase, invertase, and pepsin were not utilized at all when complexed, although they were rapidly utilized when not adsorbed.

C. Adhesion between Bacteria and Colloid Particles 1. General Consideration

Bacterial cells in clay suspension frequently adhere to clay particles, resulting in formation of flocs. The reaction may be expressed by the formula:

m B + n C = B,C,

where

B = a bacterial cell; C = a clay particle; m = number of cells; n = number of clay particles.

When the value of m or n is 1, the association should be termed a bacterium-clay complex; when both values exceed 1, the association should be termed a bacterium-clay aggregate.54

Formation of a bacterium-clay complex may essentially be discussed in terms of reactions between two particles. Generally the reaction may be determined by Brownian motion and interaction when the two particles are close together.’ However, formation of bacterium-clay aggregates may involve more complex mechanisms. The aggregate formatior! may be subjected not only to interparticle forces and Brownian motion, but also to gravitation, diffusion, and convection forces.

2, Bacterium-clay Complex Although it is not always possible to discern the

process forming bacterium-clay Complexes from that forming bacterium-clay aggregates, there are several cases in which the former process can be exclusively d i s c ~ s s e d . ~ ~ - ~ ~ Two types of the complex were recognized; that is, the BC, complex (m = 1) and the B,C complex (n = 1). Sorption of smaller particles by bacteria resulted in formation of the BC, complexes and that of bacteria by clay particles larger than cells produced the B,C complexes. The complex between bacteria and particles of bacterial size (or same effective spherical diameter) is frequently of an intermediate type (m = 1, n = 1).


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Quantitative studies show that adsorption in the BC, complex is the Langmuir types6 and in the BmC complex, the Freundlich type.S4 In the case of the intermediate-type complex, adsorption curves are distinctly S-shaped.’ ’ Reexamination of data on “one-cell, one-clay particle complex,” which was produced with cells of E cofi and pyrophyllite (or kaolinite) particles of bacterial size, indicates that the adsorption curves were also S-shaped, as shown in Figure 10.

Adsorption of the Langmuir type may indicate the existence of a range of saturation levels of adsorbed clay per unit of cell surface. LahavS5 considered that the saturation level is closely related to the orientation of clay particles on the bacterial surface. Because of their platelike structure, the particles may be oriented in three different ways: (1) face-to-face sorption with broad particle (platelet) surface facing the cell surface; ( 2 ) edge-to-face sorption with the edge of the platelets facing the surface; (3) mixed sorption with platelets sorbed in both ways.

As to the BC, complex of Rhizobium, MarshallS6 found two distinct groups: some rhizobia possessed exclusiveIy carboxyl ionogenic groups at the cell surface and others predominant- ly the acidic groups along with some basic (amino) groups. The former cells sorb approximately twice as much clay as the latter. The difference of the saturation levels between the two groups was

interpreted by distribution of carboxyl and amino groups on the cell surface and oriented clay sorption to the cell surface.

Adsorption of the Freundlich type in the B,C complex may be interpreted by change in speed of the Brownian motion and the orientation of collisions between cell and clay particles. The B,C complex with clay homoionic to Na’ ion would result from the association between the positively charged edge of clay particle and the negatively charged cell surface.54 By cell sorption to clay particle of larger size, the Brownian motion would become abnormal and the chance of effective collision, in which the edge of the clay plate effectively encounters the surface of the next cell, would gradually decrease. In the case of clay particles of bacterial size, the situation would be more striking: by “one-cell, one-clay particle” association, the motion of the complex would become extremely irregular; the chance of effective collision with the next cell would suddenly decrease. Consequently, the “one-cell, one-clay particle complex” would be observed in a wider range of cell concentrstion (adsorption curve of S-shaped).

3. Bacterium-clay Complex with Clay Retaining Di- or Trivalent Cations

Cations, especially di- or trivalent cations held on the surface of clay particles, greatly affect both

0

0 1 2 3

0

0 10 20 8 In i t i a l ce l l concentration ( x 10 particles/ml )

FIGURE 10. Adsorption of E. coli on clay particles homoionic to Na‘ ions. (A) Pyrophyllite particles with the mean diameter of ca 0.9 pm were suspended at a concentration of 6.1 X 10’ particles/ml. (From Hattori, T., unpublished data.) ( B ) Kaolinite particles with the diameter of 0.7 pm were suspended a t a concentration of 3.2 x 10” particles/ml. (Replotted from Hattori.”)

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the formation and nature of the cell-clay association, probably by two effects: ' (1) bringing the surface potential of the flat surface to a lower negative value and ( 2 ) forming chelate bonds between organic groups of cell surface and the cations on the flat surface. This concept is supported by the following facts. The adsorption capacity of clay of bacterial size is markedly increased by partial replacement of Na' ion with di- or trivalent cations, as shown in Figure 1 1. The complex thus formed shows a pH-dependent stability quite different from that with clay homoionic to Na' ion (see Section III.C.4). The bacteriumclay complex with clay adsorbing di- or trivalent cations may be denoted as the BmCM (or BCM,) complex, where CM indicates a clay particle keeping di- or trivalent cations.' 96 '

4. Factors Ajyeecting Formation or Stabilitv of Bacterium-clay Complexes

Hydrogen ion concentration is an important

factor in formation or stability of a bacterium-clay complex. Surface charge of bacterial cells and clay particles, determining largely the interactions between them, will vary with change of pH of the medium. Negative charge of cell surface wd1 decrease widely with decrease in pH, and the isoelectric point of many bacteria will be between pH 2 and 3. At pH lower than the isoelectric point, cell surface usually carries positive charges. Although the flat surface of a clay particle carries negative charges in a wide range of pH, the size of the charge will become smaller with decrease of pH, and the positive charge localized at the edge of the platelet will be observed only in an acidic solution. Consequently, the BmC complex will be expected to be very stable in an acidic solution, but unstable in an alkaline solution. The result in Figure 12B supports this expectation. It is to be noted here that at pH lower than the isoelectric point (pH 2 to 3), a larger number of cells rapidly sorbed to clay (pyrophyllite) particles. This may

0 2 4 6 8 Cell concentration ( x 10 /ml )

b'lGURE 1 1 . Adsorption of E. coli on clays treated with Cu'* or Co** ions. (a) Pyrophyllite treated with Co" ions, 9.4 x lo' particleslml. (b) Pyrophyllite treated with Cu" ions, 1.7 X 10" particleslml. (c) Kaolinite treated with Cu" ions, 1.0 X 10" particles/ml. Clays were of the same size as those in Figure 10. W'rorn Hattori, T . , J . Gen. Appl. Microbiol.. 16, 3 5 1 , 1970. With permission.)

440 CRC Critical Reviews it? Microbiology

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1.0 0.8 0 . 6 0.4 0 . 2 0 0 .2 0.4 0 .6

t- Nacti HC1 +

0.10 0 .os 0 0.0s 0.10

+ NaoH HC1 + FIGURE 12. Effect of HCI and NaOH addition (meg/I) o n formation or stability of bacterium-clay complexes. Ordinate indicates the number of cells adhered to one clay particle. (A ) E. coli-pyrophyllite, treated with Fe’+ ions, complex (From Hattori, T., unpublished data.) (B) E. coli-pyrophyllite, homoionic to Na’ ions, complex (a) and E. coli-kaolinite, homoionic to Na*, complex (b). (From Hattori, T.,J. Gen. Appl . Microbiol., 16, 351, 1970. With permission.)

be interpreted by the association between neutral- ized or positively charged cell surfaces and weakly negatively charged flat surfaces of clay. Similarly, the amount of sorbed clays per cell in the BC, complex may be larger at a moderately acidic condition than at an alkaline condition. A smaller amount of clay, however, would sorb to neutral- ized cell surface at isoelectric point or lower pH since face-to-face association would occur more f r equen t l~ . ’~ 9’

In the case of the B,CM complex, the effect of

pH is distinctly different from that with the BmC complex. The former complex is very stable in a wide range of alkaline solutions and very unstable in an acidic solution (Figure 12A), which may be attributable to the nature of chelating bonds. Salt concentration will also affect formation and stability of bacterium-clay complexes.

5. me Bacterium-clay Aggregate Based on kinetic study of reactions between

bacteria and clay (pyrophyllite) in their mixture

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suspension, three types of reactions in the aggregate formation were recognized.’ ’

1. Formation of the bacterium-clay complex without appreciable formation of the bacterium- clay aggregate. In E. coli-kaoline mixture suspension, the reaction forming the aggregate was not observed for 2 hr, although the complex formation was completed immediately after mixing of two suspension.

2 . Formation of the complex followed by formation of the aggregate. In E. coli-pyrophyllite suspension, the complexes were immediately formed after mixing. Aggregate-forming reaction proceeded gradually between the complexes themselves or the complex and clay particles. Free cells were not involved in the aggregate formation.

3. Simultaneous formation of the complex and the aggregate. In Arthrobacter simplex- pyrophyllite mixture, both reactions occurred immediately after mixing. These reactions may rather belong to “short-term” reactions. There may be various other reactions belonging to ‘‘long-term’’ reactions, some of which may result from viscous materials exudated from microorganisms.

6. Sorption of Microbial Cells to Soil Particles Sorption phenomena of microbial cell to

natural soil particles may be more complex, which may be determined by a variety of factors. For example, Zvyagintsev’ observed that cells of S. marcescence attached by their sides to most particles and, on some particles, by their pole. Beutelspacher” suggested, on the basis of an electron microscopic study of soil, that bacterial flagella may provide some means of binding cells to soil particles. The slime layer produced by microorganisms may also play an important role in the adhesion of cells with soil particles. Peele6’ showed that a positively charged fraction from soil sorbed cells of Azotubacter very strongly, but the negatively charged fraction did not sorb the bacterium. He also observed that soil saturated with Fe3+ and A13+ sorbed to the cells the strongest; soil saturated with Li’, Na’, K’, and NH; adsorbed them the least strongly.

7. Growth of Bacterial Cells in the Bacteriiimclay Complex or Aggregate

Chemical activities and growth of bacterial cells in the bacteriumclay complexes or aggregates have

not received sufficient study to discuss their characteristics from the standpoint of the current view. A preliminary study was done on growth of E. coli bacterium-clay complexes.62 Cells of E. coli adsorbed on pyrophyllite particles of bacterial size holding Fe3+ ion on the surface were grown in a salt medium with glucose in test tubes in which the E. culi-pyrophyllite aggregates were in a state of sediment. As shown in Figure 13, growth occurred mainly in the part of sediment, probably in a sorbed state; rates of growth and glucose consumption were the same with two case cultures with and without addition of the clay. These results may be consistent with the concluding remarks of earlier observations (see Section 1I.B).

D. Roles of Ciay Colloid Particles in Microbial Life in Soil

Stotzky ’ emphasized that a single environmental factor, such as the type of clay minerals present, will greatly influence microbial life in soil. Outstanding examples of such influence are the correlations between the rapid spread of Fusarium oxysporum f. cubense and Histoplasma capsula- tum in certain soils and the lack of montmorillonite-type clays in such soils. The so-called “long- life” soils of Central America, in which 17 oxysporum f. cubens fails to spread, all contain mon tmorillonite.

Clay particles also play important roles in the survival of bacteria in soil. Dommergues6 showed a protective effect of a clay: survivals of A. vinelandii, A. chroucoccum , and Beijerinckia indica at low humidity were markedly enhanced by addition of kaolinite. Marshall, after the success in overcoming survival problems of root nodule bacteria in a field situation by addition of montmorillonite to a sandy showed that addition of montmorillonite or illite to a gray sandy soil protected Rhizobium trifolii from heat treatment at 70°, but kaolinite did not. The effect was interpreted by formation of a protective envelope with the clays around each cell, thereby modifying the rates of water movement into and out of the cells during drying and rewetting.’ >6

According to Miyamoto,6 soil-borne viruses survive for a long period of time by sorption to clay particles. Montmorillonite also appears to protect microorganisms against hypertonic osmotic pressure. Microbial activity is depiessed at a higher salt concentration, but the depression is lessened by addition of montrnorill~nite.~’


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T i m (hours)

1-IGURE 1 3 . Growth of E. coli in bacterium- pyrophyllite, treated with Fe”, aggregates. Closed circles indicate growth of cells in the supernatant and opened ones growth of cells in the aggregates. The estimation of the latter was carried with the suspension, in which bacterium-clay aggregates were decomposed and cells were fully dispersed by adding HCI and by shaking. Numbers indicated by opened circles show those of the suspension after being dispersed. Dotted line shows growth of the bacterium in the same liquid medium without clay with the same amount of innoculum. (From Hattori, T. and Hattori, R., in Biseibutsu no Seitai (Microbial Ecology), Vol. 3 , University of Tokyo Press, Tokyo, in press. With permission.)

The most important role of clay or soil colloid particles in microbial life in soil is that they have an ability to make up the structure of soil aggregates. This is of paramount significance in relation to physiology and ecology of soil microorganisms.

IV. SOIL AGGREGATES

One of the essential characteristics of soil microhabitat is its discreteness: the chemical, physical, and biological characteristics of the microhabitat differ widely from point to point or from time to time. This concept is based on the fact that soil is a heterogeneous, discontinuous, and structured environment, composed of soil particles of various sizes.

From the discreteness of soil microhabitat, Stotzky’ derived three tentative concepts.

1. This discontinuity and variability in particle size may result in soil being a composite of enumerable microbial communities, each circumscribed by its own immediate environment.

2 . If the concept of discrete microhabitat is accepted, even if only temporarily, it is possible to accept the concept of the diversity of microhabitats and, therefore, the variability in the microbial composition between even closely adjacent microhabitats.

3. A further assumption to be derived from the microhabitat concept is that, because of the relatively small size of the microhabitats and their essential isolation from one another, only small, environmental changes may be necessary to alter their microbiological composition. Although soil as a whole is generally considered to be a “well-buffered system” in “dynamic equilibrium,” the site of the microhabitats may be less buffered and undergo rapid and extreme fluctuations.

In this section it will be shown that the discrete soil microhabitats can be essentially characterized at the level of soil aggregate, and microbiological principles can be extended to the core realm of microbial life through understanding of the structure and characteristics of the habitats.

A. Physical Constitution of Soil The physical consitution of a soil material is

determined by the size, shape, and arrangement of the solid particles and voids, including both the primary particles to form compound particles and the compound particles themselves. The primary particles are, according to their size, termed clay colloidal particles, silt particles, and sand. Col- loidal soil particles have a tendency to stick together and the reaction is coagulation.

Coagulation of soil colloids is accelerated by

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the presence of cations. When soil particles are coagulated by monovalent ions, coagulation may be reversible. On the other hand, aggregated particles by trivalent cations is irreversible. Coagu- lation by bivalent cations occupies an intermediate position with respect to its reversibility. Colloid particles can also be coagulated by simple collisions, if the colliding particles carry charges of opposite signs. Aggregates thus formed still have coarse structure.

An aggregate formed by primary particles alone is called the primary aggregate; that formed by the primary aggregate is the secondary one, and so on. Tyulin6 ' recognized two groups of microaggregate below 0.01 mm in diameter. The first group consists mainly of microaggregates formed with humic substances and from humic substances themselves, coagulated by calcium ions. The second group is microaggregates coagulated by sesquioxides, mainly by iron ion.

Formation of macrostructure (larger than 0.01 mm) is closely related to its origin from the primary particles or microaggregates. Factors involved in formation of macroaggregates may include the change in its volume on moistening, drying, freezing, and thawing; pressure of plant systems; activities of burrowing and digging animals; actions of microorganisms; and soil- loosening agricultural implements.

The porosity of soil may depend mainly on the mutual arrangement of solid particles in soil mass. The entire variety of pores occurring in natural soil may be explained by a certain packing of soil particles.

B. Internal Structure of Soil Aggregates 1 . Pore Size Distribution and Moisture Retention of Soil Aggrega res

The size and distribution of pores in soil are among the most important factors determining microbial life since microorganisms live exclusively in pore space, and retention of water indispensable to microbial life is closely related to the size of each pore. A great difference is recognized between physical conditions of water in culture medium and in soil. The former can be considered to be continuous and homogeneous at least macro- scopically, but the latter cannot. The physical condition of soil water is described approximately in terms of matrix potential. The principal forces which contribute to the soil water potential (a measure of the free energy status of water) are

those associated with the soil matrix, those associated with the osmotic characteristics of the soil solution, and those which affect the total pressure on the soil water. As an approximation, we can make an assumption that pore space in soil aggregates consists mainly of capillary tubes of various size and that, among the forces, the capillary force is of paramount importance for a range of water content. Thus, the water potential is approximated by the matrix potential, \k,, which is defined as

(26)

where

M = molecular weight of the liquid; p/po = relative vapor pressure; V = specific volume of the liquid; u = surface tension of the liquid; r = radius of curvature of the capillary

pore.

This energy may be expressed logarithmically by the pF scale;6' that is,

pt; = lop 'Pm (27)

A further approximation can be made,

PI: = log 0.15 - log r (28)

where r is expressed in centimeters6 We can estimate the pore size distribution of

soil aggregates from a differential water content- pF curve. However, it should be noted here that soil pores are not of uniform diameter, but rather vase-shaped with abundant cross linkages. In a drying soil the capillary radius determining the potential is the radius of the pore necks rather than that within the enlarged section of the pore. On the other hand, when wetting occurs, water enters the system in gushes; the critical radius and the potential are related to the widest portion of each pore's segment rather than to the pore neck.

2. Fine Structure of Soil Aggregates Emerson" presented a physical aspect of the

microstructure of a soil aggregate. Assuming that a soil aggregate is composed mainly of clay and quartz. these particles may be linked directly by

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organic matter or through two or more domains which are themselves linked by organic matter. Most clay domains are groups of oriented clay particles. Some clay domains may also be held together by coulombic attraction between a positive edge and a negative face. In normal agricultural soils, where calcium is the dominant exchangeable cation, clay particles in the aggregate may be aggregated into clay domains by drying. Drying to the wilting point may be sufficient. This model suggests a heterogeneous composition of internal surface of pores in soil aggregates (Figure 14A).

In a soil aggregate there may be three types of pores, as shown in Figure 14B: tubular pores with narrow opened ends, “bottle” pores, and closed pores. Water in closed or partially closed pores may not move as freely as that in opened pores and, thus, may have a considerably higher pF value as compared with that in opened pores of the same size. Opened pores may be connected with narrower or constricted tubes, and the connecting system may be of three dimensions.

Most humic substances are in a state linked wi th mine ra l subs t ances . According to Kononova,” the following forms of linkage between humic substances and the mineral part of soil are recognized.

1. Humic substances interact with cations of

alkali and alkaliearth metals, in which active sites are carboxyl and phenolic hydroxyl groups.

2. Humic substances interact on the surface of mineral particles with hydroxides of aluminum and iron occurring in the colloidal state. The complexes of alumino- and ferrohumic gels thus formed do not peptize in water and firmly fix to the mineral particles.

3. Humic acids (and probably fulvic acids) and their derivatives become firmly held on the surface of the crystal lzttice of the minerals by the action of van der Waals’ forces and hydrogen bands.

4. Fulvic acids and some forms of humic acids form complex compounds with Fe, Al, Mn, Cu, Zn, and certain other elements.

Organic substances including humic substances are frequently sorbed and surrounded by clay particles or other minerals or are shut up in closed pores. Such organic substances may be protected from microbial decomposition since microbial cells or their exoenzymes would not come in contact with the organic substances. When the microstructure is destroyed by mechanical force or freezing of moist aggregates followed by thawing, microbial cells can enter and more easily attack the organic compounds.

/ - \ quartz organic matter c l a y domain

(1) ( 2 ) ( 3 )

FIGURE 14. ( A ) Pores surrounded by clay domain, quartz domain, and organic matter domain. (B) Various types of pores - ( 1 ) closed pore; (2) bottle-type pore; and ( 3 ) tubular pore with open ends.

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C. Soil Aggregates as Microhabitats I. Bacterial Cells Retained by Soil Aggregates of Various Size

It is generally believed that most bacterial cells in soil are attached to the surface of soil particles and rarely occur in soil solution. Presumably taking this belief into account, some authors consider bacterial cells to be distributed uniformly or randomly in a soil aggregate. On the other hand, one may expect from the concept of the discrete microhabitats of soil microorganisms that bacterial cells might distribute nonrandomly, forming clumps. In approaching the distribution pattern of bacterial cells in soil aggregates, bacterial cells retained by soil particles will be discussed in relation to their size both quantitatively and qualitatively.

Yamagishi7 * obtained various size fractions of soil particles by sedimentation velocity and, after washing out free-form cells, resuspended them in sterilized water. Particle number and particle-size distribution were obtained by Coulter counter (Model A) and bacterial number by the plate

method. I f necessary, to enumerate the number of cells clumped in soil aggregates, soil particles (aggregates) were disrupted by sonic oscillation at 10 kc for 3 min. As shown in Figure 15, percentage of soil particles retaining cells increased in proportion to the mean effective diameter with the particles smaller than ca. 10 pm in diameter. This suggests that bacterial cells may occur randomly on the surface of soil particles; the probability of occurrence of cells may be proportional to the size of surface area.

A marked increase in bacterial number per particle was observed with soil particles of size ranging ca. 10 to 20 pm in diameter. This may be interpreted only by assuming that bacteria may be able to live more abundantly or multiply in inner parts of the aggregate larger than 1Opm in diameter. Inside the aggregate there may be pores large enough to admit bacterial cells, since the probability of occurrence of cells on the unit outside the surface of aggregates may be considered to be constant with aggregates of a wide range of size. This was shown by Zvyagintsev, who

L

0 1 2 3

Diameter of soi l part ic le ( logarithms )

FIGURE 15. Mean number of cells retained by one soil particle or aggregate as a function of particle size. Diameter is expressed in micrometers. (Calculated from data of Yamagishi.’ * ) (From Hattori, T., Microbial Life in the Soil, Marcel Dekker, New York, 1973. Courtesy of Marcel Dekker, Inc.)


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used reflected illumination to count cells sorbed on soil particles.’ If this assumption is the case, bacterial cells are neither uniformly nor randomly distributed, but clumped, particularly inside pores of the aggregate.

Yamagishi’s experiment” presented further evidence suggesting random distribution of bacterial cells on soil particles less than ca. 10 pm in diameter and clumped distribution inside soil particles (aggregates) larger than 10 pm. She isolated about 50 strains from each soil particle fraction (6 and 16 pm in the mean diameter) and from each of two soil particles (370 and 430 pm in diameter). She classified these bacteria based on their morphological and physiological characteristics (Figure 16). It should be noted in Figure 16 that microbial composition was of the greatest variety with the particles of 6 pm. With larger particles, the cumber of isolates probably belonging to the same or related species had a

tendency to increase. The result suggests again that although various bacteria may distribute randomly on the outside surface of soil particles, probably in a state of nongrowing under a field condition, bacteria in capillary pores inside larger particles (aggregate) may be able to metabolize substances more actively and sometimes succeed in multiplication. Thus, in the inner pores, cells of similar characteristics may be clumped.

2. Two Types of Microhabitats in a Soil Aggregate We can more reasonably develop the concept of

soil aggregate as microhabitats of bacteria on the basis of water retention in each site of the aggregate. As mentioned in Section IV.B.1, the amount of water present may depend largely on the size of the pores. Pores can be divided into two groups according to their water retention: ( 1 ) noncapillary or large pores which do not hold water tightly and ( 2 ) capillary or small pores

h b e r of isolates

FIGURE 16. Bacterial composition of soil aggregates of various size. A and B indicate bacterial compositions of soil particle fractions with the mean diameter of 6 and 16 pm. C or D indicates the composition of a soil aggregate of the mean diameter of 374 or 432 pm. White columns indicate Grampositive nonspore former, dotted columns Gram-positive spore former, and striped columns Gram-negative bacteria. Each square shows bacteria belonging to the same or very similar species. (Modified from Yamagishi, H., Ph.D. thesis, Tohoku University, Sendai, Japan, 1968.)

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which hold water tightly. The noncapillary pores drain freely after rain or irrigation; on the other hand, the capillary pores contain water which remains even after most free drainage is completed (at field capacity). Retention of capillary water is considered to be between pF 2.7 to 4.5, which corresponds to pores of ca. 6.0 to 0.1 ym in diameter. Considering size of bacterial cells, pores of 2 to 6 pm in diameter may be the most favorable microhabitats of soil bacteria. Although soil particles less than 10 pm may rarely possess such capillary pores, those larger than 10 ym frequently do, and there may be many capillary pores in soil aggregates larger than ca. 20 ym. The clumped distribution of bacterial cells may be interpreted by such capillary pores.

The concept of a capillary pore as the most favorable microhabitat of soil bacteria is also supported by several observations. Seifert73 observed that nitrification was stimulated by increase of water content until pF 2.7 and realized a maximum rate; further increase of water content did not result in stimulation of the activity. A similar result was also obtained by Moser and Olson74 on sulfur oxidation. Tanaka7' showed tha t by using two soils (Noken loam soil and Kitagama sandy loam soil), the number of bacteria increased proportionally to water content of soil until a critical value, which was specific to each soil, of water content was realized. With a larger amount of water than the value, the number was not affected distinctly by water content (Figure 17). It is to be noted here that, although the critical .value was specific to each soil, pF value corresponding to the critical value was between ca. 2.7 to 3.0.

These results support the concept that the capillary pores are more favorable microhabitats for bacteria. As to PO or inverse effect (nitrification and sulfur oxidation were depressed by water lower than pF ca. 2 ) of increase in water observed with soils retaining a larger amount of water than that at pF ca. 2.7, there may be three possible interpretations. (1) When large noncapillary pores are saturated with water, oxygen diffusion into soil aggregate may become very difficult; consequently, bacterial activity and growth may be markedly depressed. (2) Soluble substances in large pores may be eluted by rain or irrigation; thus, nutrients indispensable for bacterial growth may be apt to be wanting. (3) Some protozoa eating bacteria may be able to move and attack

their prey in larger pores since the mean diameter is assumed to be 5 /-un for flagellates, 10 pm for amoebae, and 20 ym for cilliates.

The second and third statements suggest that noncapillary pores, probably larger than 6 pm, may not be as favorable microhabitats as capillary pores, even when water is sufficiently supplied. At field condition, bacterial cells cannot behave actively most of the time in those pores.

We may conclude that in a soil aggregate there would be two types of microhabitats: capillary pores which are more favorable for bacterial life and noncapillary pores and the outside surface of

" 'A - A n 7

a a

0 10 2 0 30 411

I latcr content (\I

4 '

0 5 10

IVater content (9.1

FIGURE 17. Relationship between bacterial number and water content of soil. (A) and (B) indicate Noken loam soil and Kitagama sandy soil, respectively. Opened and closed circles indicate numbers of Gram-negative and Gram-positive bacteria, respectively. (From Tanaka, H. and Sakamoto, M., Bull. Inst. Agric. Res. Tohoku Univ., 2 3 , 141, 1972. With permission.)


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the aggregate which are not favorable. The former may be called the inner part or region of a soil aggregate and the latter the outer part or region.

3. Microbial Composition and Variation in the Inner and the Outer Part of a Soil Aggregate

Based on the concept of the differentiated microhabitats of a soil aggregate, one may expect that differences in bacterial compositions would frequently be observed between two populations in the inner and the outer part of a soil aggregate. To test this, soil aggregates are mildly shaken in sterilized water to disrupt cementing bonds between macroaggregates, by the arrangement of which large noncapillary pores are formed; thus, bacterial cells in the outer part may be dispersed into the liquid. After cells thus dispersed are washed out, soil aggregates are disrupted by sonic oscillation; thus, bacterial cells in the inner part may be dispersed into water.7 7 3 7 ’

Although this washing-sonication technique does not offer a rigid criterion of the cells in the inner and outer regions of soil aggregates, it was shown by several experiment^'^ that the technique is very useful as an approximation to microbial cells in the differentiated microhabitats. For example, the two bacterial fractions showed different responses to antiseptic chemicals. When soil aggregates were put into 10-4M HgClz solution, bacterial number of the cell fraction of the outer region decreased rapidly, although the number of the other fraction decreased gradually. This indicates that HgZ + ions diffused more easily to cells in the former fraction as compared to those in the latter fraction.’’

By means of this technique, distributions of various bacteria were examined in terms of those in the inner and outer parts (Figure 18). It should be noted that ratio of number of Gram-negative bacteria to total bacteria, a t field conditions, was

A

n

B c D E FIGURE 18. Microbial flora in the inner and the outer region of soil aggregates. Number of total bacteria was taken as 100 arbitrarily in each case. White columns indicate number of each microbial group in the outer part and striped columns that in the inner part of soil aggregates. A, 8 , C, D, and E indicate Gram-negative bacteria, spore form cells of bacteria, bacteria resistant to 10% NaCI, actinomy- cetes, and fungi, respectively. (Calculated from data of Hattori.19 )

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usually as small as or smaller than 10% in the outer part; on the contrary, the ratio was much higher and frequently exceeded 30% in the inner part. Most bacterial cells in the outer part were in spore form, although smaller parts were in spore form in the inner part. Halotolerant bacteria and nitrifying bacteria were also more abundant in the inner part. Such differences in bacterial compositions between the inner and the outer parts may be closely related to water and salt content of each microhabitat.

Although little is known about distribution of fungi, it appears that fungi are more abundant in the outer part, probably owing to the size of their spores or hyphae and to bacterial a n t a g ~ n i s m . ~ ~ It was also noted that some fungi such as Fusarium were constantly found in the inner fraction but not in the outer fraction. Other fungi such as Aspergillus were found mainly in the outer fraction.* These results may suggest some specific associations between fungi and soil components.

4. Bacterial Growth iii Soil Aggregates Generally, bacterial number in soil aggregates is

considered to be largely limited by water and nutrient supply. Soil aggregates at field or air-dried conditions are wanting in water, especially in the outer part. I f nutrient solution is supplied to the

9 h

E A 2 u

v 3 .i

5 7 a0 \ m

aggregates, flourishing growth of bacteria may occur in both microhabitats.8 ’ Figure 19 shows that bacterial growth occurred when soil was percolated with glycine solution. Greater increase in bacterial number was observed in the outer part. The maximal number in each microhabitat may be determined by the volume of available space of each as well as other factors. In the case of Tsukisappu volcanic ash soil with larger volume of capillary pores as compared with that of Taketoyo soil (used in the experiment of Figure 19), the maximal number in the outer region was less than that in the inner region.

In the case of Figure 19, bacterial number increased by ca. 4,000 times in the outer and ca. 30 times in the inner part during 3 days percolation. If there was one bacterial cell in each pore and all of the progeny cells were admitted in the same pore, the volume of each pore should be larger than 10,000 pm3 in the outer and 60 pm3 in the inner part (one cell was assumed to occupy a volume of 2 pm3). The volumes may correspond to ca. 27 and 5 pm in diameter, respectively. In the case of the outer part, this assumption that bacterial cells were distributed randomly in pores of ca. 27 pm in diameter may not be unreasonable as an approximation for two reasons: (1) Zvyagintsev’s observation7 (see Section 1V.C. 1)

r

$ 0 && 0 5 10 1 5

Time ( days )

FIGURE 19. Bacterial growth in soil aggregates percolated witH glycine solution. Opened and closed circles indicate numbers of bacteria in the outer and the inner part of soil aggregates. (From Nioh, T. and Furusaka, C., J. Sci. Soil Munure (Japan), 40, 149, 1969. With permission.)


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and ( 2 ) total volume of pores with diameter of 27 pm may be 10,000 X lo5 pm3 (number of cells in the outer is 10s/g-soil before percolation) and corresponds to several percent of volume of noncapillary pores in this soil. (In the experiment soil aggregates of 2 t o 4 mm in diameter were used.) The possibility of bacterial growth on the outside surface of the aggregates will be discussed later.

In the case of cells in the inner part, however, the above assumption may not be reasonable for the following reasons. (1) Cells are expected to be clumped in capillary pores (as discussed in Section IV.C.l and again later). If this is the case, the volume of the pores should be greater by several times. (2) The above calculation for volume necessary to admit cells in each pore is under- estimated for the space necessary for bacterial life. Corrected volume may be too large for a capillary pore. Consequently, we would try to make another assumption on bacterial growth in the inner part. In a capillary pore there may live several cells before percolation; if soil aggregates are percolated with glycine, cells will multiply in the pore until the pore space will be saturated with cells (probably a pore of 4 pm in diameter will be saturated with about 10 cells). I f the pore is opened with tubes wide enough for bacterial cells to pass, cells will disperse into another pore by movement or elongation of divided cell chain and grow until the new microhabitat is saturated. By such a dispersion mechanism, cells in one and the same capillary pore before percolation will grow until 10 to 20 vacant pores will be saturated with newly divided cells. The total volume of capillary pores thus occupied by newly divided cells may correspond to only several percent of total volume of capillary pores or microhabitats in the inner part of the aggregates.

5. State of Bacterial Cells in Soil Aggregates As to the state of bacterial cells in soil

aggregates, there are two problems to be discussed here: (1) whether or not all cells are sorbed on the surface of soil particles and (2) whether or not cells in pores, especially those in capillary pores in the inner part, are in a “trapped” state.

Generally it is accepted that most of the bacteria that occur in soil are attached t o soil particles and d o not occur in large numbers in soil solution. Based on this concept, Gray et al.’ considered that it would seem more realistic to

calculate the percentage of the surface area that bacterial cells colonize than to estimate the relative volume occupied by microbial cells. However, in real soil free or sorbed state of bacterial cells is not always rigid or fixed, but flexible and sometimes interconvertible.

Although it is very difficult to distinguish sorbed cells from free cells in the strict sense of the terminology, we can approach this by applying the dependency of settling or centrifuging velocity on particle density and radius. Nioh and Furusakaa3 tried to study bacterial state in soil aggregates by means of centrifugation in a two- layered sucrose solution system. Figure 20b gives the partitions of purely cultured cells of E. coli and B. sztbtilis into each fraction, and Figures 20c and d give those of bacterial cells in the inner and the outer parts. As a tentative criterion, cells in fractions A, AB, and B will be considered to be in a “free” state and those in fractions BC, C, and D in a “sorbed” state. In this experiment it may be probable that some of the cells attached to soil particles would be detached by shaking soil suspension and especially (in the case of cells in the inner part) by sonication. However, cells firmly sorbed on a surface may not be desorbed by this procedure, as shown in the case of E. coli cells sorbed on an anion exchanger, Dowex By disruption of soil aggregate, most of the sorbed cells would be dispersed into liquid medium, and some of the cells would be sorbing fragments of soil particle.

In air-dried aggregates most cells in the outer part were in the “sorbed” state; in the inner part, however, about 30% of the cells were in the “free” state. When soil aggregates were subjected to percolation with glycine solution and bacterial number increased to a maximum, percentage of cells in the “free” state increased rapidly both in the inner and the outer part. After I S days of percolation, bacterial number decreased gradually and percentage of cells in the “free” state also had a tendency to decrease.

These results suggest that a t dried or semidried conditions, most of the cells in the outer part may be in the “sorbed” state; on the other hand, those in the inner part may not be necessarily so. Some of cells in the inner part may be in the “free” state, probably owing to the fact that effective internal surface of capillary pores may frequently be occupied by cells. When bacteria grow in soil aggregates, cells may multiply at first on internal

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100

80

60

40

20

0 A A B B B C C D

A A B B B C C D

.~~

A AB B BC C D

A A B B B C C D

Fractions

FIGURE 20. Fractionation of bacteria in soil aggregates in two-layered sucrose solution system by centrifugation. The whole system was centrifuged at 750 X g for 15 min. (a) Position of each fraction. (b) Distribution of E. coli (opened circles) and B. subtilis (triangles) in pure cultures. (c) Distribution of bacteria in the outer part of soil aggregates percolated with glycine for 1 hr (opened circles) and 3 days (closed circles). (d) Distribution of bacteria in the inner part of the aggregates percolated for 1 hr (opened circles) and 3 days (closed drcles). (From Nioh, T. and Furusaka, C., Soil Sci. Plant Nufr. (Tokyo), 18, 219, 1972. With permission.)

surface of pores both in the inner and the outer part. After having occupied the effective area of the surface, newly divided cells may be detached from the surface, as discussed in Section II.E.2. As to the outside surface of soil aggregates, cell growth may not be as flourishing as that in pores (probably owing to deficiency of salt nutrient), and divided cells may frequently be attacked by protozoa or eluted out by percolated liquid.

As to the second problem, Allison84 considered that microorganisms are too large to penetrate a stable aggregate, and some bacterial cells may be trapped inside when the aggregate is formed. Such organisms would probably find conditions for growth unsatisfactory; if they did grow, cell growth would soon split the aggregate. The concept of “trapped bacteria” may reflect in part the situation of bacterial’ cells living in soil aggregate.

The concept may be supported by the following evidences: (1) existence of a great variety of bacteria, most of which may probably dwell separately as single or several species-mixed cultures in different pores and thus survive without microbial antagonism and (2) the persistence of organic compounds in the aggregate. However, some bacteria in the inner part may not be trapped rigidly in closed pores; if water is supplied sufficiently, they become locomotive from pore to pore by Brown motion, flagella motion, water movement, or cell divisions followed by elongation of newly divided cell-chains. On the other hand, most noncapillary pores may be opened, and cells may easily enter into the pore, but may not be washed out by perfusate liquid since they may be protected by bottle neck. Only bacterial cells on the outside surface of the aggregate may be


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washed out by the liquid, if they are not in a sorbed state.

It may be also noted here that exogenously added cells can penetrate both the outer and the inner regions of the aggregate with a partition ~ o e f f i c i e n t . ~

6. Oxygen Diffusion into Soil Aggregates The life of microorganisms in soil aggregates is,

to a great extent , affected or restricted by the availability of oxygen. The rate of oxygen diffusion into soil aggregates is determined by: (1) the size of specific surface area, through which oxygen enters the structure; ( 2 ) the difference in concentration between that at the surface and that in the inner part; (3) the geometry of the pathways for oxygen diffusion; and (4) diffusion coefficient of the medium. Seifert” showed that the intensity of nitrification is a linear function of specific surface area, which was interpreted in terms of factor 1.

A theory for the distribution of oxygen in soil aggregates was developed in relation to microbial life by Greenwood.’ ?’’ Based on an assumption that microbial cells would be distributed uniformly in the aggregates and metabolism would switch from aerobic to anaerobic pathways when oxygen concentration drops below approximately 3 X 1 0 - 6 M , he derived a n equation determining the size of anaerobic sphere, in which respiration is assumed to cease:

where

R , = radius of the oxygen-free sphere; Vo = volume of the aggregate; V l = volume of the oxygen-free sphere; D = diffusion coefficient; C, = oxygen concentration around the

Q = rate of oxygen uptake per volume. aggregate;

in real soil aggregates, however, there may be extremely irregular pathways, having very close necks, for gas diffusion; thus, the anaerobic zone is expected to occur more frequently than predicted by Equation 29. The widespread distribution of obligate anaerobes and the simultaneous occurrence of nitrification and denitrification in soil support this expectation.

The diffusion and concentration of carbon dioxide and other volatile chemicals produced by microorganisms may also influence more or less the life of microorganisms in soil aggregates. Recently, Smith’ ’ noticed that ethylene is produced by spore-forming bacteria and not only causes soil fungistasis but also regulates the activity of most soil microorganisms.

7. Growth of Nitrifving Bacteria in Soil Aggregates Lees and Quastel’ 3 ’ noted that growth of

nitrifying bacteria would be closely related t o soil crumbs and presented a concept of “soil saturated with nitrifying bacteria.” Recently, Nishio and Furusaka’ 7’ 9-9 made a careful study of the kinetics and numbers o f nitrifying bacteria, e s p e c ia I 1 y n i t r i te-oxidizing bacteria during repercolation of nitrite. The main results were

1 . When soil aggregates were percolated with ammonium salt or nitrite solution, nitrifying bacteria grew more rapidly in the outer part than the inner part of soil aggregates.

2 . Number of nitrite-oxidizing bacteria increased logarithmically, though rate of the oxidation became seemingly constant.

3. When soil aggregates were dispersed, about 50% of ammonium oxidizing bacteria were sedimented in a minute and most of nitrite- oxidizing bacteria in 30 sec in the case of air-dried soil. Both bacteria had a tendency to be sedimented more slowly as soil percolation was continued.

4. With air-dried soil there was no observation of any appreciable effect of sonic oscillation on number of nitrifying bacteria in soil suspension

,prepared by hand shaking, bu t the effect became apparent as percolation was continued.

5. Rate of nitrite oxidation with soil percolated by nitrite was increased by disruption of soil aggregates. Ratio of rate with intact aggregates (vp) to that with disrupted ones (V,) decreased with duration of percolation.

6. Nitrite was oxidized exponentially in the early stage of nitrite percolation and in “cube root relation throughout the percolations.”

7. There was no appreciable difference between values of Michaelis constant for nitrite oxidation with intact and disrupted soil aggregates.

Although the question o f microbial growth during reperfusion is not easy t o handle mathe-

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matically,g2 we would try to interpret these results on the basis of the concept of the discrete microhabitats in soil aggregates. We will make two additional assumptions. (1) Nitrifying bacteria in soil aggregates may grow exclusively on some specific soild surface (specific site) exuding sticky substances; thus, the cells may be sorbed so firmly on the surface that we cannot disperse them even by sonic oscillation. The growth may be ceased when the site is covered by the bacteria or other bacteria or when microenvironment directly circumscribing the site is changed unfavorably. ( 2 ) There may be a chance that some cells will detach from the site and settle on other sites. The probability may be proportional not directly to number of cells on each site, but to the number of sites themselves covered with the cells.

The first assumption may interpret the dis- crepancy between nitrifying activity and number of nitrifying bacteria; the activity with disrupted aggregates should be proportional to number of cells, but not to that of the sites (the value obtained as “number of nitrifying bacteria”). Consequently, we can estimate the mean cell number of various sites from the activity and the number of the sites. Assuming from data of Laudelout et al.q3 that the amount of nitrite oxidation per cell per hour is 3 X lO-’NO;-N pg/hr, the mean number of nitrite-oxidizing bacteria per each site is calculated from data of Nishio and Furusakaa9 (Table 3 ) . The number of sites growing .the bacteria did not increase appreciably during the first percolation with nitrite and since the second percolation, showed an exponen- tial increase, although rates of nitrification became seemingly constant. We can interpret this based on

Table 3. Nitrite-oxidizing bacteria in air-dried soil may survive mainly on the site of larger area (several hundred pm’) because the probability of finding surviving cells may be larger; during the first percolation the bacteria may grow mainly on the site. Since the second percolation, cells may settle to other vacant sites. The consideration is consistent with Nishio and Furusaka’s results (3,4, and 6). In the case of ammonium-oxidizing bacteria, the situation may be similar to nitrite- oxidizing ones, except that the maximum number of cells per each site may be less than 1/10 the value of nitrite-oxidizing ones as the specific activity is higher with the former than the latterq3 and observed cell number (number of the site) was larger.

As to the second assumption, newly settled sites may usually be small in size in the effective area (less than 10 pm2) and number of the sites may increase logarithmically. It may occur fte- quently that several sites are bound together so tightly that their dispersion by sonic oscillation is effective. This consideration is also consistent with results 2, 3, and 4.

I t may be expected, as in the case of glycine percolated soil aggregates, that bacterial growth in response to the nutrient in perfusate may occur more immediately in the outer part of the aggregates than in the inner part. Oxygen diffusion into the inner part may frequently be a limiting factor for bacterial activity. This may interpret results 1, 5 , and 7.

8. Microbial Interactions in Soil Aggregates I t may be noted that microbial interactions are

also developed on the stage of the aggregate

TABLE 3

Calculations of Mean Numbers of Nitrite-Oxidizing Bacteria on Each “Site”

Oxidation rate of nitrite per “site”

The most probable number in Soil aggregate (number of sites growing “dispersed state”* Mean cell number at the end of the bacteria) per g soil NO, -N pg/hr per each site?

x 104 2nd percolation 48.4 5th percolation 204 7th percolation 4,000

x lo-’ 10.0 333

3.3 110 0.25 a

Note: Soil was percolated with 10 mM sodium nitrite solution. *The rate was estimated from the v p Y , relation. tRate of nitrite oxidation per cell was assumed to be 3 X lo-’ NO, -N pglcell/hr. Based on data of Nishio and Furusaka.”’


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structure. In the inner part the dominant inhabi- tants are bacteria, although some fungi also exist. Although the inner part is a more favorable microhabitat for bacteria, the cells are not so crowded that close interactions between cells of various species always occur. Small cell clumps are scattered very thinly. Percentage of capillary pores settled by bacteria may be less than 0.1 at air dried or field conditions and less than 10 even at ,a condition saturated with bacteria by percolation of glycine solution. On the other hand, fungi and some protozoa, as

well as bacteria, can act vigorously in the outer part of soil aggregates if water and nutrients are sufficiently supplied. As to interaction between bacteria and fungi in the outer part, an interesting fact was presented by Cook and P a p e n d i ~ k . ~ ~ Microbial response to water content with different soils can be described generally in terms of the matrix potential, although the response occurred a t different water contents with different soils. Number of bacteria in soil aggregates increased in response to the amount of added mixture of glucose and ammonium at a potential pF 3 or less. Number of germ tubes of a fungi (Fusaliiim roseum), which exist preferentially in the aggregates, lysed or converted new chlamydospores resistant to bacterial attack at a potential pF 4 or less, whereas at higher potential germlings branched and appeared to grow. This suggests that at a lower potential than pF 4 bacteria will be able to grow actively in response to increase of water content in larger pores in which the germing fungus spores exist. The fungus will lyse probably owing to bacterial attack. On the other hand, at a higher potential, bacteria may be unable to grow in these pores since bacteria are more sensitive than fungi to low moisture; thus, the fungal germ may be able to grow without bacterial attack.

We may consider that at a field condition microhabitats in the outer region of soil aggregates will be favorable to fungi and not to bacteria; on the contrary, microhabitats in the inner region will be favorable only to bacteria.

D. Dynamic Feature of Microbial Populations in Soil Aggregates

Microbial populations in soil aggregates are essentially not in a static state but in a dynamic state, which may involve two equilibria; that is, the equilibrium between various populations in soil aggregate and that through formation and

decomposition of soil aggregates. The former equilibrium may be closely related to differentia- tion of microhabitats into those in the inner part (capillary pores), those in the outer part (noncapillary pores and the external surface of the aggregate), and also to discrete structure of the microhabitats in the inner part (equilibrium between c and d in Figure 21). These considerations may probably be in harmony with Stotzky’s concepts. As to the latter equilibrium, i t should be mentioned that soil particles to be incorporated into the aggregate may retain bacterial cells at a probability proportional to the surface area, as mentioned in Section IV.C.l. Such cells may frequently be trapped inside the aggregate and thus survive more persistently. On the other hand, bacterial cells proliferated in response to various nutrients may be released from the microhabitat when the aggregate is decomposed. Cells in a free state may lose their viability more frequently than those in a sorbed state. Such dynamic equilibrium is illustrated in Figure 21.

Formation and decomposition of soil aggregate are aIso believed to be closely related to microbial actions. Undoubtedly, this may also offer attractive problems from our standpoint. However, little is known to discuss as yet.

V. SOIL PROFILE

A. Soil Profile as a Macroscopic Environment of Microorganisms

From a standpoint of microbial habitats, we recognize a vertical face of soil having a characteristic layered pattern. Each individual layer is known as a horizon, and the set of layers is called a soil profile. Horizons can be grouped into upper, middle, and lower, according to the position they occupy. Those in the upper position occur at or near the surface, and microorganisms act most vigorously in it. Usually they contain the largest amount of organic substances, and in humid areas the greatest amount of water passes through them. The middle position includes those horizons in which microbial activity is not as high as in the upper. The lower position is occupied by relatively unaltered materials, and microbial activity is the lowest in those horizons.

The growth and activity of microorganisms in soil may, globally, be considered to be closely connected to mass flow in it. Consequently, we should discuss mass flow in soil in relation to

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0 0 1

n

FIGURE 21. A cyclic change of microhabitats of bacteria in relation to soil aggregates. (a) Random distribution of bacteria on soil particles. Bacteria in a free state may be rare. (b) Intermediary microhabitats in soil aggregate just formed. (c) Microhabitats in soil aggregate at a field condition. (d) Bacteria are enriched in soil aggregate by addition of nutrient solution. When either water or nutrient disappears, the microhabitats may return again to (c). (e) Decomposed aggregate in which cells in a free state may be killed more rapidly under unfavorable conditions.

microbial life in the last chapter. Study of microbial reaction in soil profile was carried out by means of a continuous flow system by Macura and Malek" Recently, both theoretical and experimental simulations of the reaction were developed by M ~ L a r e n ~ ~ and Bazin and S a ~ n d e r s . ~ '

B. Simulation of Microbial Reactions in Soil Profiles by Small Glass Beads System

Bazin and Saunders developed theoretical aspects of nitrification in a small glass bead column based on McLaren's concept of vectorial reactions. They considered microbial reaction in the column as a continuous flow system with a dilution rate, D. D is equal to -j,,/V, where j, is the flow rate and V i s the void volume. Based on an assumption that the nitrifying bacteria in the column are at a steady state with respect to distance down the cdumn, they derived equations to evaluate the steady state of the sequential reaction NHd -, NO; -+ NO; :

dS, /dt = DS, - DS, - brn, (30)

dS,/dt = k, m, - DS, - Am, (32)

(33) dm,/dt = Am, - f, (m,)

dS, /dt = k, rn, - DS, (34)

where So = input concentration of ammonium in

S1 = output concentration of ammonium in

Sz = output concentration of nitrite in

S3 = output concentration of nitrate in

m, = density of Nitrosomonas in p d m l N; mz = density of Nitrobacter in pg/mI N ; p = specific growth rate of Nitrosomonas; X = specific growth rate of Nitrobacter; kl = reaction constant for nitrite forma-

pg/ml NH;-N;

pg/ml NH;-N;

,ug/ml NO;-N;

pg/ml NO; -N;

tion;

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kz = reaction constant for nitrate forma-

D = rate of washout -jo/V, where jo is the tion;

flow rate and V is the void volume.

These equations describe “change in ammonium c o n ce n t rat ion ,” “change in Nitrosomonas, ” “change in nitrite,” “change in Nitrobacter,” and “change in nitrate.”

They showed experimentally that these equations well fitted the result by using:

f , = k, Drnt , f, = k, Drni (35)

where k3 and are constant:;. These equations are equivalent to assume that growth of the organisms can be described by a modification of the Verhulst-Pearl logistic equation.” In their second report’ they tried to generalize reaction equations suggesting some processes establishing nonuniform distribution of nitrifying bacteria be formulated.

C. Temporal and Vectorial Characteristics of Nitrifying Reactions in Soil Profile

Gases and water in soil are not standing, but always diffusing and moving. The movements of these materials are important factors in supporting and connecting microbial activities in different sites of soil aggregates or soil profile. Taking this mass flow in soil into account, McLaren’ O 0

emphasized that microbial reactions in soil are both temporal and vectorial.

In soil water-soluble substances, such as ammonia (either produced by microbial action or added exogenously), are subjected to a generally downward movement so that consecutive reactions of ammonium in soil are expressed as a function of depth (x) as well as time (t). Equations determining concentrations of Nb’, NO;, and NO; in a soil enriched by continuous flow were derived; that is

(NO-) = -~__ K’ (NH‘)o [exp(-K, X) - exp(-K, X)] K2 - K,

(37 )

where K1 and K2 are constant and X is the distance from the top of the column. Suffix 0 refers to the initial concentration.’ McLaren also obtained equations for soils in which nitrifying bacteria are growing.’ ’ These formulations were essentially supported by both column and field experiments.

The above discussion is valid only in the absence of ion exchange reactions, unless the system is in a steady state.104a Moreover, it must be mentioned that other microorganisms and plant roots in soil will take up some of each nutrient, and only a fraction of the initial ammonium may reach lower depths. In the absence of plants, however, the equations work well.’ O 4 J 0 4 ’ Never- theless, the concept of vectorial reactions may be of great value in approaching microbial life in soil from the standpoint of microbial kinetics.

VI. SOME OTHER PHYSICAL ENVIRONMENTAL FACTORS

In this presentation our attention has been focused on several physical environmental factors which are necessary for our attempt to extend principles of microbiology to soil microorganisms. Discussion of these factors has been done in a very restricted sense. For, example, we have assumed that the water potential can be approximated by the matrix potential, but the state of soil water at a high pF value may not be fully understood in terms of capillary forces. I t may also be an essential problem to examine whether or not both the matrix potential and the osmotic potential affect microorganisms similarly.

It should be noted here that most other physical factors may also be closely related to the physical constitution of soil depicted in this presentation. For example, salt concentration is known to largely affect microbial growth; the words “halophilic,” “halotolerant ,” and %on- halophilic” refer to organisms which are favorably, more or less indifferently, and unfavorably influenced, respectively, by certain concentrations of salt in their aqueous environment. ZoBell showed that in soil there are various types of bacteria in relation to their growth response to salt concentration from 0.5% to 30%.’05 The present authors noticed various types of bacteria sensitive to salt concentration in varying degrees as well as those tolerant t o salt, as shown in Figure 22. Coexist- ence of such bacteria may be also interpreted by

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100

0 0.01 0.1 0 . 5 1 2 5 10 1 5

Concentration of NaCl (I$)

F1,GURE 22. Various types of growth response to salt concentration, which were observed among isolates from a paddy soil. Isolates without star mark were tested in one 100th dilution of the usual nutrient broth. Isolates with star mark were grown in the usual broth. Abscissa is in logarithmic scale. (From Hattori, R. and Hattori, T., unpublished data.)

the discrete and discontinuous microhabitats in soil aggregates. Most halosensitive bacteria were highly sensitive to various organic substances.

Temperature also affects microbial growth and activity. The effect may not be the same between bacteria in liquid medium and in soil since bacteria sorbed on a surface showed different temperature dependency of their growth and activity! ' 3 4 4

VII. CONCLUSION

Studies of microbiology have been based largely on the proposition that growth is the expression par excellence of the dynamic nature of living organisms, and the kinetic aspect has been a very useful tool in approaching the growth.' O 6 J O 7

Unfortunately, soil environments are too complex

to receive kinetic studies of microbiologists. In this presentation we attempted to describe the complex soil environment in a simpler way so that kinetic studies of microbial growth can be extended to those in soil. Taking account of the paramount importance of the physical environment of soil, we tried to reconstruct the environment in a stepwise way by introducing several levels of physical concepts (such as solid surface, colloidal particles, soil aggregate, and soil profile) to show that microbial growth at each level can be studied on the basis of kinetics. We also showed that these physical concepts involve many basic problems to be considered in relation to microbial growth as well as their ecological behaviors. The future of soil microbiology will depend very largely on the study of these problems.


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REFERENCES

1 . Stotzky, G., Activity, ecology. and population dynamics of microorganisms in soil, in Microbial Ecology, Laskin, A. I . and Lechevalier, H., Eds., CRC Press, Cleveland, 1974,57.

2. Hattori, T., Microbial Life in the Soil, Marcel Dekker, New York, 1973. 3. Waksman, S. A., Soil Microbiology, Chapman & Hall, London, 1952. 4. Alexander, M., Introduction to SoilMicrobiology, John Wiley & Sons, New York, 1961. 5 . Gray, T. R. G., Bwby, P., Hill, I. R., and Goodfellow, M., Direct observation of bacteria in soil, in The Ecology of

Soil Bacteria, Gray,T. R. G. and Parkinson, D., Eds., Liverpool University Press, Liverpool, 1968, 171. 6. Marshall, K. C., Sorptive interactions between soil particles and microorganisms, in Soil Biochemistry. Vol. 2,

McLaren, A . D. and Skujins, J . J . , Eds., Marcel Dekker, New York, 1971. 7. Zvyagintsev, D., Interaction between Microorganisms and Solid Surfaces, Moscow University Press, Moscow, 1973. 8. Karpinskaya, N. S., O n the problem of sorption of bacteria on soil, Nauchino Agron. J., 9,587, 1926. 9. Chudiakov, N. N., Ueber die Adsorption der Bakterien durch den Boden und den Einfluss derselben auf die

mikrobiologischen Bodenprozesse, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt . 2, 68, 345, 1926. 10. Peek, T. C., Adsorption of bacteria by soils, Cornell Univ. Agric. Exp. Sta. Memoir, p. 197, 1936. 1 1 . Dianow, E. B. and Woroshilowa, A. A., Sorption of bacteria on soil and its effect on microbial activity, Nauchino

Agron. J. 9,520,1925. 12. Sohngen, N. L., Einfluss von Kolloiden auf mikrobiologische Prozesse, Zentralbl. Bakteriol. Parasitenkd.

Infektionskr. Hyg. Abt . 2, 38, 621, 1913. 1 3 . Rubentschik, L., Roisin, M . B., and Bieijansky, F. M., Adsorption of bacteria in salt lakes, J. Bacteriol., 32, 1 1 ,

1936. 14. Conn, H. J . and Conn, J . E., The stimulating effect of colloids upon the growth of certain bacteria, J. Bacteriol., 39,

99, 1940. 15 . McCafla, T. M., Physico-chemical behavior of soil bacteria in relation to the soil colloid,J. Bacteriol., 40, 33, 1940. 16. Lees, H. and Quastel, J. H., Kinetics of, and the effects of poisons on, soil nitrification, as studied by a soil perfusion

technique, Biochem. J . . 40, 803. 1946. 17. Lees, H. and Quastel, 3. H., The site of soil nitrification, Biochim. J . , 40, 815, 1946. 18. ZoBell, C. E. and Anderson, Q., Observations on the multiplication of bacteria in different volumes of storcd sea

water and the influence of oxygen tension and solid surfaces, Biol. Bull., 71,324, 1936. 19. ZoBell, C. E., The effect of solid surfaces upon bacterial activity, J. Bucteriol., 46, 39, 1943. 20. Heukelekian, H. and Heller, A., Relation between food concentration and surface for bacterial growth, J. Bacferiol.,

40,547, 1940. 21. Poindexter, J . S., Biological properties and classification of the Caulobacter group, Bacteriol. Rev., 28,231, 1964. 22. Herbert, D., Elsworth, R., and Telling, R. C., The continuous culture of bacteria; a theoretical and experimental

study, J. Cen. Microbiol., 14,601, 1956. 23. Larsen, D. H. and Dimmick, R. L., Attachment and growth of bacteria on surfaces of continuous-culture vessels, J.

Bacteriol., 88, 1380, 1964. 24. Topiwala, H. H. and Hamer, G., Effect of wall growth in steady-state continuous culture, Biotechnol. Bioeng., 8,

919, 1971. 25. Helmstetter, C. E. and Uretz, R. B., X-ray and ultraviolet sensitivity of synchronously dividing E. coli, Biophys. J..

3,35 ,1963. 26. Helmstetter, C. E. and Cummings, D. J., Bacterial synchronization b y selection of cells a t division, Proc. Narl. Acad.

Sci. U.S.A., 50,767, 1963. 27. Helmstetter, C. E., Rate of DNA synthesis during the division cycle of E. coli B/r, J. Mol. Biol., 24, 417, 1967. 28. McLaren, A. D. and Skujins, J., The physical environment of microorganisms in soil, in The Ecology o f Soil

Bacteria, Gray, T. R. G. and Parkinson, D., Eds., Liverpool University Press, Liverpool, 1968, 3. 29. Goldman, R., Goldstein, L., and Katchalski, E., Water-insoluble enzyme derivatives and artificial enzyme

membranes, in Biochemical Aspects ofReuctions on Solid Supports, Stark, G . R., Ed., Academic Press, New York, 1971 , l . Hartley, G. S. and Roe, J. W., Ionic concentrations of surfaces, Trans. Faraday SOC., 36,101. 1940. McLaren, A. D. and Babcock, K. L., Some characteristics of enzyme reactions at surfaces, in Subcellular Particles. Hayashi, T . , Ed., Ronald Press, New York, 1959. McLaren, A. D. and Skujins, J., Nitrification by Nitrobacrer agilis on surfaces and in soil with respect to hydrogen ion concentration, Can. J . Microbiol., 9, 729, 1963. Marshall, K. C., Stout, R., and Mitchell, R., Mechanism of the initial events in the sorption of marine bacteria to surfaces,J. G e n Microbiol., 68, 337, 1971. Marshall, K. C. and Cruickshank, R. H., Cell surface hydrophobicity and the orientation of certain bacteria at interfaces, Arch. Mikrobiol., 91,29 , 1973. Hogg, R., Healy, T. W., and Fuerstenau, D. W., Mutual coagulation o f colloidal dispersions, Trans. Furaday sac., 62, 1938,1966.

30. 31.

32.

33.

34.

35.

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36,

37. 38.

39, 40. 41. 42.

43.

44.

45.

46.

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48.

49. 50.

51.

52.

53. 54. 55. 56.

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58. 59.

60. 61.

62.

63.

64.

65.

66.

67. 68. 69. 70. 71. 72.

73. 74.

Hattori, R., Physiological Characteristics of Bacteria Sorbed on a Surface, Ph.D. thesis, Tohoku University, Sendai, Japan, 1968. Pethica, B. A., The physical chemistry of cell adhesion, Exp. Cell Res. Suppl., 8 , 123, 1961. Marshall, K. C., Stout, R., and Mitchell, R., Selective sorption of bacteria from sea water, Can. J. Microbiol., 17, 1413,1971. Weisq L., The Cell Periphery, Metastasis and Other Contact Phenomena, North-Holland, Amsterdam, 1967. Hattori, T. and Furusaka, C., Chemical activities of E. coli adsorbed on a resin, J. Biochem. (Tokyo), 48,831, 1960. Hattori, R. and Hattori, T., Effect of a liquid-solid interface on the life of microorganisms, Ecol. Rev., 16, 63, 1963. Hattori, T. and Furusaka, C., Chemical activities of Azotobacter as'lis adsorbed on a resin, J. Biochem. (Tokyo), 50, 312,1961. Munson, R. J., Turbidostats, in Methods in Microbiology, Norris, J. R . and Ribbons, D. W., Eds., Academic Press, London, 1970,349. Hattori, R., Hattori, T., and Furusaka, C., Growth of bacteria on the surface of anionexchange resin. 1. Experiment with batch culture, J. Gen Appl. Microbiol., 18, 271, 1972. Hattori, R., Hattori, T., and Furusaka, C., Growth of bacteria on the surface of anionexchange resin. 2. Electron microscopic observation of adsorbed celIs growing on resin surface by carbon replica method, J. Gen. Appl. Microbiol.. 18, 285, 1972. Hatton, T. and Furusaka, C., Chemical activities of E. coli adsorbed on a resin, Biochim. Biophys. Acf4. 31, 581, 1959. Hattori, R., Growth of E. coli on the surface of an anion-exchange resin in continuous flow system, J. Gen. Appl. Microbiol.. 18, 319, 1972. Dawes, I. W. and Thornley, J. H. M., Sporulation in Bacillus subtilis. Theoretical and experimental studies in continuous culture system, J. Gen. Microbiol.. 62,49 , 1970. Hattori, R., Spore formation of B. subtilis adsorbed on the surface of an anion exchange resin, to be published. Stotzky, G . and Rem, L. T., Influence of clay minerals on microorganisms. I . MontmoriIlonite and kaolinite on bacteria, Can. J . Microbiol., 12, 547, 1966. Stotzky, G., Influence of clay minerals on microorganisms. 111. Effect of particle size, a t i o n exchange capacity, and surface area on bacteria, Can. J. Microbiol., 12, 1235, 1966. Stotzky, G. and Rem, L. T., Influence of clay minerals on microorganisms. IV. Montmorillonite and kaolinite on fungi, Can. J . Microbiol.,' 13, 1535, 1967. McLaren, A. D., Use of mole fractions in enzyme kinetics, Arch. Biochem. Biophys., 97, 1, 1962. Hattori, T., Adhesion between cells of E. coli and clay particles, J . Gen. Appl. Microbiol., 16, 351, 1970. Lahav, N., Adsorption of sodium bentonite particles on Bacillus subtilis, Plan[ Soil, 17, 191, 1962. Marshall, K. C., Interaction between colloidal montmorillonite and cells of Rhizobium species with different ionogenic surfaces, Biochim. Biophys. Acta, 156, 179, 1968. Krishnamurti, K. and Soman, S. V., Studies in the absorption of bacteria. Part 1. Adsorption of B. subtilis and B. coli by kaolin and charcoal, Proc. Indian Acad. Sci. Sect. B , p, 81, 195 1. Hattori, T., Interactions between bacteria and c l ~ y s , to be published. Beuterspacher, H., Wechselwirkung zwischen anorganischen Kolloiden des Bodens, 2. Pfinzenernaehr. Dueng. Bodenkd., 69, 108, 1955. Peele, T. C., Adsorption of bacteria by soils, Cornell Univ. Agric. Exp. Sta. Memoir, p. 197, 1936. Santoro, T. and Stotzky, G., Influence of cations on flocculation of clay minerals as determined by the electrical sensizing zone particle analyzer, Soil Sci. SOC. Am. Proc., 31, 761, 1967. Hattori, T. and Hattori, R., Microbial growth on a solid-liquid interface, in Biseibufsu no Seitai (Microbial Ecology), Vol. 3, University of Tokyo Press, Tokyo, in press. Dommergues, Y., Study of some factors influencing the behavior of the soil microflora during the process of drying, Sci. Sol, p. 141, 1964. Marshall, K. C. and Rogers, F. J., Influence of fine particle material on survival of Rhizobiurn trifolii in sandy soil, Nature, 198,410, 1963. Marshall, K. C., Mechanism of the initial events in the sorption of marine bacteria to surfaces, J. Gen. Microbiol., 68, 337, 1971. Miyamoto, Y., Further evidence for the longevity of soil-born plant viruses adsorbed o n soil particles, Virology, 9, 290, 1959. Tyulin, A. F., Soil structure in forests, Pochvovedenie, 30, 1954; 1, 33, 1955. Schofield, R. K., The pF of the water in soil, Trans. 3rd Int. Congr. Soil Sci., 2, 37, 1935. Smart, P., Soil microstructure, Soil. Sci., 119, 385, 1975. Emerson, W. W., The structure of soil crumbs,J. Soil Sci , 10, 235,1959. Kononova, M. M., Soil Organic Matter, Pergamon Press, Oxford, 196 1. Yamagishi, H., Interrelation Between bacteria and Colloid Particles, Ph.D. thesis, Tohoku University, Sendai, Japan, 1968. Seifert, J., The ecology of soil microbes, Acta Llniv. olrol., 3, 245, 1965. Moser, U. S. and Olson, R. V., Sulfur oxidations in four soils as influenced by soil moisture tension and sulfur bacteria, Soil Sci.. 76, 251, 1953.


Cri

tical

Rev

iew

s in

Mic

robi

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

ifor

nia

San

Fran

cisc

o on

11/

20/1

4Fo

r pe

rson

al u

se o

nly.

75.

76.

77.

79. 78.

ao .

82. 83.

84. 8s.

87.

88. 89.

81.

86.

90.

91.

92.

93.

94.

95.

96. 97.

98. 99.

100. 101. 102.

103.

104.

Tanaka, H. and Sakamoto, M., Studies on soil physical conditions and the ecology of soil-borne disease bacteria. 4. The relationships between bacterial numbers and either soil water potential (pF) or moisture contents, Bull, Inst. Agric. Res. Tohoku Univ., 23, 141, 1972. Stout, J. D. and Heal, 0. W., Protozoa, in Soilffiology, Burges, A . and Raw, F., Eds., Academic Press, London, 1967. Hattori, T., Bacterial distribution and variation in soil aggregates, J. Sci. Soil Manure (Japan), 37, 32, 1966. Hattori, T., Fractionation of bacteria in soil aggregates, Soil Biol., 11, 30, 1969. Hattori, T., Microorganisms and soil aggregates as their microhabitat, Bull. Inst. Agric. Res. Tohoku Univ., 18, 159, 1967. Nishio, M. and Furusaka, C., The distribution of nitrifying bacteria in soil aggregates, Soil Sci. Plant Nutr. (Tokyo), 16, 24, 1970. Nioh, T. and Furusaka, C., Studies on glycine-percolated soil. 11.. J. Sci. Soil Manure (Japan). 40, 149, 1969. Sato, K., Bacteriological studies on percolated soil, Bull. Inst. Agric. Res. Tohoku Univ., 22, 93, 1971. Nioh, T. and Furusaka, C., Studies of glycine-percolated soil. IV. Fractionation of bacteria in glycine-percolated soil in two layered sucrose solution system, Soil Sci. Plant Nutr. (Tokyo), 18, 219, 1972. Allison, F. E., Soil Organic Matter and its Role in Crop Production, Elsevier, Amsterdam, 1973. Seifert, J., Influence of the size of soil structural aggregates on the degree of nitrification, Fol. Microbiol., 9, 115, 1964. Greenwood, D. J . and Berry,G., Aerobic respiration in soil crumbs,Narure, 195, 161, 1962. Greenwood, D. J., The effect of oxygen concentration on decomposition of organic materials in soil, Plant Soil, 14, 360, 1961. Smith, A. M., Ethylene: a cause of fungistasis, Nature, 246, 311, 1973. Nishio, M. and Furusaka, C., The number of nitrite-oxidizing bacteria in soil percolated with nitrite, Soil Sci. Plant Nutr. (Tokyo), 17 ,54 , 1971. Nishio, M. and Furusaka, C., Kinetic study of soil percolated with nitrite, Soil Sci. Plant Nutr. (Tokyo), 17, 61, 1971. Nishio, M. and Furusaka, C., Number of nitrifying bacteria retained by soil particles of various size, J. Soil Sci. Manure (Japan), 40, 315, 1969. Paul, E. A. and McLaren, A. D., Biochemistry of the soil system, in Soil Biochemistry, Vol. 3, Paul, E. A. and McLaren, A . D., Eds, Marcel Dekker, New York, 1975. Laudelout, H., Simonart, P. C., and van Droogenbroeck, R., Calorimetric measurement of free energy utilization of Nitrosomonas and Nitrooacter. Arch. Mikrobiol., 63, 256, 1968. Cook, R. J. and Papendick, R. I., Soil water potential as a factor in the ecology of Fusarium roseurn f. sp. cereah “culmorum.”PlonrSoil, 32, 131, 1970. Macura, J . and Malek, I., Continuous flow method for the study of microbial processes in soil samples, Nature, 182, 1796, 1958. McLaren, A. D.,Temporal and vectorial reactions of nitrogen in soil: a review, Can. J . Soil Sci.. 50,91 , 1970. Bazin, M. J. and Saunders, P. T., Dynamics of nitrification in a continuous flow system, Soil Biol. Biochem., 5 , 53 1, 1973. Pielou, E. C., A n Introduction ro Mathematical Ecology, Interscience, New York, 1969. Saunden, P. T. and Bazin, M. J., Nonsteady state studies of nitrification in soil: Theoretical considerations, Soil Biol. Biochem.. 5,545, 1913. McLaren, A. D.,Nitrification in soil: System approaching a steady state, Soil Sci. Soc. Am. ROC., 33,55 1, 1969. McLaren, A. D., Kinetics of nitrification in soil: Growth of the nitrifiers, SoilSci. SOC. Am. Proc.. 35,91, 1971. Ardnkani, M. S., Rehbock, J. T., and McLaren, A. D., Oxidation of nitrite in a soil column, Soil Sci. Soc. Am. Proc., 37,53, 1973. Ardakani, M. S., Rehbock, J. T., and McLaren, A. D., Oxidation of ammonium to nitrate in a soil column, Soil Sci. Soc. Am. Proc., 38,96,1974. Ardakani, M. S., Schultz, R. K., and McLaren, A. D., A kinetic study and nitrite oxidation in a soil field plot, Soil Sci. SOC. Am. Proc.. 38, 273. 1974.

104a. Ardakani, M. S., Volz, M. G., and McLaren, A. D., Consecutive steady state reactions of urea, ammonium, and nitrite nitrogen in soi1.J. SoilSci., 55, 83, 1975.

105. ZoBeU, C. E.,il.IarineMicrobiology, Chronica Botanica, Waltham, Mass., 1946. 106. Van Niel, C. B., The kinetics of growth of microorganisms, in The Chemistry and Physiology of Growth, Parpart, A.

K., Ed., Princeton University Press, 1949, 91. 107. Monod, J., The growth of bacterial cultures, Annu. Rev. Microbiol.. 3, 371, 1949. 108. McLaren, A. D., personal communication. 109. Doner, H. and McLaren, A. D., personal communication.

May 1976 461

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