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From Winogradsky's column to contemporary research using bacterial microcosms


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The fast pace of scientific research often means that contemporary investigators are unaware of critical developments resulting from the work of previous generations of scientists. We were superficially aware of Sergei Winogradsky (1856 – 1953) through his columns, currently in teaching to illustrate aspects of microbial succession and community function, but were unaware of his importance as the founder of microbial ecology and the first to use microcosms to study bacterial physiology. Here we take the opportunity to remind fellow microbiologists of his work, presenting in homage columns constructed using water and sediments from the Dnipro (Dnieper) collected close to his place of birth. We then provide a review of our own research using microcosms to investigate aspects of plant-bacterial interactions, bacterial evolution, biofilm-formation, and soil colonization, as well as recent advances in developing plastic and transparent soils to investigate root development and fungal hyphae invasion in soil pore networks, to show that more than one hundred years after the invention of the microcosm, they are still being used profitably in microbial research. A PDF of this is available on request to AS.
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In: Microcosms ISBN: 978-1-62618-661-3
Editor: Christopher C. Harris © 2013 Nova Science Publishers, Inc.
Chapter 1
Olena Moshynets1, Mariia Boretska2 and Andrew J. Spiers3*
1Laboratory of Microbial Ecology, Institute of Molecular Biology and Genetics,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
2Department of General and Soil Microbiology,
D. K. Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
3The SIMBIOS Centre and School of Contemporary Sciences,
University of Abertay Dundee, Dundee, UK
The fast pace of scientific research often means that contemporary investigators are
unaware of critical developments resulting from the work of previous generations of
scientists. We were superficially aware of Sergei Winogradsky (1856 1953) through his
columns, currently in teaching to illustrate aspects of microbial succession and
community function, but were unaware of his importance as the founder of microbial
ecology and the first to use microcosms to study bacterial physiology. Here we take the
opportunity to remind fellow microbiologists of his work, presenting in homage columns
constructed using water and sediments from the Dnipro (Dnieper) collected close to his
place of birth. We then provide a review of our own research using microcosms to
investigate aspects of plant-bacterial interactions, bacterial evolution, biofilm-formation,
and soil colonization, as well as recent advances in developing plastic and transparent
soils to investigate root development and fungal hyphae invasion in soil pore networks, to
show that more than one hundred years after the invention of the microcosm, they are still
being used profitably in microbial research.
* E-mail address:
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Olena Moshynets, Mariia Boretska and Andrew J. Spiers
The science of medical microbiology was established through the pioneering work of
scientists such as Louis Pasteur (1822 1895), Ferdinand Cohn (1828 1898), Heinrich
Koch (1843 1910), and others in the last part of the 19th century. Pasteur made significant
breakthroughs investigating the cause and prevention of diseases, Cohn instigated bacterial
classification based on morphology, and Koch became famous for his postulates defining
pathogens. During this period, the early isolation and characterisation of bacteria relied on the
use of universal growth media and the presumption that all microbial activity was the result of
specific isolates functioning alone. In contrast, the pioneering work of the environmental and
ecological microbiologists, Martinus Beijerinck (1851 1931) and Sergei Winogradsky
(1856 1953), are often overlooked and are perhaps unfairly overshadowed by their
contemporaries who studied human disease. Beijerinck and Winogradsky investigated the
biochemical role bacteria play in important natural processes, including nitrification, nitrogen
fixation and sulfur
oxidation. Winogradsky emphasised repeatedly that the objectives of
ecological microbiology differed from those of medical microbiology [109, 110]. He
pioneered the use of the selective
approach to study biological processes in contrast to the
universal medium used by Koch and others to isolate and characterise pure strains (see the
quote from Winogradsky [105, 106] in [109]), as well as the realisation that in order to
understand some biological processes, communities of bacteria needed to be studied rather
than single cultures. Winogradsky is often described as the father of microbial ecology, and is
considered the founder of contemporary ecological microbiology in Russia where his
approach led to the Winogradsky School of Microbiology
[2, 17, 109, 110]. More relevant to
this book is that Winogradsky was one of the first to use and promote microcosms in
microbial research.
Sergei Nikolaevich Winogradsky
was born in 1856 into a wealthy family in Kyiv (Kiev),
then part of Russia (a photograph of him is presented in Figure 1). He appeared to have had a
troubled education, selling his golden graduation medal from the Second Kyiv Gymnasium (a
secondary or grammar school) in 1873 in response to what he felt was an uninteresting,
disagreeable, physically and morally humiliating style of teaching’ (Selam Waksman, quoted
in [17]). He entered the University of Kyiv in 1873 to study law following his brother and
father, but transferred to the natural sciences within a month before leaving the university
altogether. He spent a year at the St. Petersburg Academy of Music studying piano, before
finally entering the University of St. Petersburg in 1877 to study the natural sciences and
microbial physiology with Andrey Famintsyn.
We adopt the spelling convention for sulfur and sulfur compounds as recommended by the Royal Society of
Chemistry Nomenclature Committee.
Winogradsky referred to this as ‘elective’.
In comparison, Beijernck’s Delft School of Microbiology was relatively well-known to non-Soviet
microbiologists and historians of biology.
Also transliterated from the Russian (Виноградский) or Ukrainian (Виноградський) as ‘Vinogradskii’.
From Winogradsky's Column to Contemporary Research ...
Figure 1. S.N. Winogradsky and soil landscapes. (Left) Photograph of Winogradsky (c. 1936). (Right)
Winogradsky drew ‘soil landscapes’ to show the relationship between soil microorganisms and
particles (Brie soil sample, ~100 µm wide; c. 1924). Images are from Winogradsky [105] and Seliber
An outline of his impressive professional career is provided in the table at the end of this
Chapter, and several relevant areas of his work are described in this section (the details
summarised here are compiled from various references including [1, 2, 3, 17, 75, 78, 82, 104,
109, 110]; we do not cite Winogradsky’s research publications here as they can be accessed
through the references provided). In Famintsyn’s laboratory, Winogradsky examined the
nutritional and physiological properties of the yeast, Candida vini, originally known as
Mycoderma vini
. C. vini is a pathogen of sugar beet (Beta vulgaris), which was causing
problems for the developing European sugar industry, and it is also an etiological agent of
wine illness or mould. Winogradsky used Geissler chambers to investigate the growth of C.
vini cultures under different conditions. Detailed descriptions of this apparatus originally used
by Pasteur [61] and its use by Winogradsky can be found in [2, 17]. In his biography of
Winogradsky, Waksman suggests that these investigations of C. vini are the first careful
investigations ever made on the influence of controlled environment on the growth of
microorganisms in pure culture, under well-defined experimental conditions’ (Waksman,
quoted in [17]). They may also be the first significant use of microcosms in the study of
microorganisms, and Famintsyn’s approach of coupling nutritional and physiological
experiments with microscopic observations became Winogradsky’s modus operandi [17].
Having completed his Master of Science degree at the University of St. Petersburg,
Winogradsky joined Anton DeBary at the University of Strasburg in 1885 where he studied
the filamentous sulfur-oxidizing bacterium Beggiatoa
. He was initially assigned a project to
determine whether microorganisms were monomorphic or pleomorphic
. Winogradsky chose
to investigate this using Beggiatoa, but could not prove the case that it was monomorphic as
he was unable to isolate sufficiently pure cultures from sulfur springs in Switzerland and
Germany. However, he became interested in the ability of Beggiatoa to accumulate
intracellular sulfur granules and the role of sulfur metabolism in the growth of this bacterium.
For a description of the Candida see [86].
For a description of the Beggiatoa see [98].
At the time there was a significant debate as to whether microorganisms exhibited multiple forms (i.e.
pleomorphic) or whether they only had one form (i.e. monomorphic) (now the current view). However, to
prove monomorphism, pure cultures where required for investigation.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
During this period Winogradsky also reconfigured his technical repertoire … (adding) new
variations of slide microcultures and, most important, artificial environments’ to investigate
bacteria in their natural state, allowing him to bring the ‘field into his laboratory’ [2].
Winogradsky demonstrated that the Beggiatoa oxidized H2S (hydrogen sulfide) to S0
(sulfur) to release energy and formed the sulfur granules
as a result of that process, referring
to this form of metabolism as ‘inorgoxidation’ (the oxidation of inorganic compounds). By
observing the behaviour of Beggiatoa cells under a cover-slip, Winogradsky was able to
demonstrate that the cells migrated to a narrow interface where both H2S and O2 (oxygen) co-
exist, O2 being required for the oxidation of H2S. This was also an early demonstration that
motile organisms will choose an optimal position along chemical gradients where the most
efficient metabolism and rapid growth is possible. A corollary of this is that simple
microcosms with two chemical gradients can provide a habitat choice to which organisms can
respond. From his studies of the oxidation of H2S by Beggiatoa, Winogradsky formulated the
theory of chemolithotrophy in which inorganic substrates, usually of mineral origin, are used
to obtain reducing equivalents for biosynthesis or respiration. Many chemolithotrophic
pathways are now known in which a wide range of inorganic substances, such as H2S, NH3
(ammonia), Fe2+ (ferrous iron), etc., are used by bacteria as a source of energy. For a range of
reviews on this subject, see [23, 24, 28, 39, 42, 85]. Winogradsky then moved to the Swiss
Polytechnic Institute in Zurich in 1888 where he developed his physical chemistry and
bacterial skills in association with Ernst Schultz and Otto Roth (Winogradsky had a very poor
opinion of Roth, see the quote in [17]). At Zurich, he studied the chemolithotrophy of sulfur
and iron bacterium. Winogradsky also began his monumental studies on nitrification that
provided clear evidence for the process of bacterial autotrophy and for its roles in the cycles
of Nature[17] for which he is most famously remembered. He was the first to isolate and
characterize nitrifying bacteria that oxidized NO2- (nitrite) to NO3- (nitrate), and confirmed
that the oxidation of nitrogen in soil was a two-step biological process: NH4+ (ammonium) to
NO3- and NO3- to NO2- (nitrite), with each step being performed by different organisms. This
finding led directly to the concept of sulfur and nitrogen cycling in nature. For recent reviews
on nutrient cycling and the roles of microorganisms, see [11, 20, 22, 24, 47, 49, 76, 85].
Winogradsky also reported the first example of chemoautrophy in which the energy
gained from NO2- oxidation is used to fix CO2 (carbon dioxide) to make organic compounds
for growth or energy. The significance of this was that it was the first demonstration as an
alternative to photosynthesis in which the energy from light is used to fix CO2 (for a
description of the different modes of energy conversion used by bacteria, see [79]). Dworkin
[17] suggests that at this stage in the 1890’s, Winogradsky began to recognize the need to re-
examine and reinterpret laboratory observations with what actually happens in soils and
elsewhere, marking the true beginning of the field of microbial ecology. It is noteworthy that
Ernst Haeckel first defined ecology (œkologie) in 1868 as the science of all the relations of
organisms with the external world, with the organic and anorganic conditions of existence;
… the mutual relations of all organisms living in one and the same place, their adaptation to
environmental circumstances, their transformations in the struggle for life …’ (Haeckel,
quoted by [4]). Winogradsky returned to St. Petersburg in 1891 to be the Head of the General
Microbiology Department of the Imperial State Institute of Experimental Medicine,
He also determined that S0 was further oxidized to SO42- (sulfate) to release even more energy. The SO42- is
excreted as H2SO4 (sulfuric acid).
From Winogradsky's Column to Contemporary Research ...
eventually becoming the Director in 1902. During this period he made a number of important
discoveries, including the first isolation of the free-living anaerobic nitrogen fixer,
Clostridium pasteurianum, originally known as C. pastorianum. This anaerobic bacterium
could grow under N2 (nitrogen) gas but not under normal aerobic (oxygenated) conditions. He
subsequently showed that his original isolation of C. pasteurianum in flasks was successful
because he had purified it in the presence of two contaminating aerobic organisms. These
were able to reduce O2 levels sufficiently to allow C. pasteurianum to grow anaerobically. In
soils, the co-existence of different species having different nutritional requirements and
activities underpins community function, and once again, Winogradsky’s experiments with
simple microcosms demonstrates that important ecological interactions can be identified and
experimentally tested. In 1896 he presented a lecture at the Institute of Experimental
Medicine On the role of microbes in the general cycle of life’ in which he visualized the
activity of microbes as regulating the circulation of chemicals between the organic and
inorganic realms throughout the world [2].
In 1905 Winogradsky resigned as Director of the Imperial State Institute of Experimental
Medicine and retired to his estate in Gorodok in the Ukraine. However, he was forced to flee
in the aftermath of the Russian Revolution in 1921. He spent the last thirty years of his life at
the Pasteur Institute, which at that time was the centre of European microbiology,
formulating the ideas and precepts of what was to become known as microbial ecology’ [17].
Central to this was the ‘Direct’ method which included microscopy and microphotography to
gain direct access to the microscopic landscapes of soils[2]. In Figure 1 we present a soil
landscape drawn by Winogradsky published in the 1920’s
showing the distribution of
microorganisms and particles of soil in a sample obtained from the Brie region of France
[105, 106] (his soil landscapes have an artistic appeal that are lacking modern soil images, cf.
Figures 5, 6 and 7, but in turn, his images lack the quantitative detail expected in
contemporary research). During this period, another early pioneer of soil microbiology
working in Kyiv, Nikolai Cholodny, developed the famous buried glass slide method to
sample soil microorganisms whilst maintaining some spatial detail [12, 13]. He also realized
that soil microorganisms could be studied by microscopy under natural conditions, using
small transparent soil chambers where the influence of soil treatments could be studied
experimentally. The innovative aspect of Winogradsky’s Direct method, including
contributions from others such as Cholodny, was that it did not rely on in vitro cultures but
rather allowed investigation of microorganisms directly in the complexity of their natural
environment. Edward Russell working at the Rothamsted Agricultural Experimental Station
in the UK
corresponded with Winogradsky whom he called the founder of soil
microbiology’, discussing the application of the Direct method in their work. Russell’s
colleague, Henry Thornton, began to combine it with their statistical approach to investigating
soil microbiology. However, to link microbial distributions in soils with biochemical
processes, Thornton maintained that there was still an essential place for the investigation of
in vitro cultures in soil microbiology [2], a view that was ultimately accepted by
Winogradsky in the development of his Direct method. During his career, Winogradsky was
transformed from plant physiologist, to microbiologist and then to the first ecological
It is worth mentioning that the first 35mm camera produced by Leica was not available until 1925, so hand-drawn
microscopic observations were routinely used at the time of Winogradsky’s soil studies.
Apropos nothing at all, Andrew Spiers has a link with Rothamsted as some relatives live nearby.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
microbiologist [3, 17]. In 1949 he published Microbiologie du Sol: Problèmes et Méthodes,
Cinquante ans de Recherches (Soil Microbiology: Problems and Methods, Fifty Years of
Investigations) [105, 106], a compendium of his research which included an essay describing
the synthesis of microbiology and ecology into the new discipline of microbial ecology, and
the development of soil microbiology. In this, his direct method had evolved into a synthetic
approach that accommodated the biological, chemical and physical characteristics of soils. He
argued that to study soil microbiology, a combination of techniques were required and that
the biological, physical and chemical components should be thought of as parts of a single
system [2]. In his biography Waksman concludes, in a somewhat understated manner, that
Winogradsky had made ‘significant contributions to the sciences of plant physiology,
microbiology, soil science, and ecology’ and that ‘his work unites these fields in a novel way
[104]. Winogradsky published his last paper in 1953 and died the following year aged 97.
Winogradsky began his observations of Candida vini using Geissler chambers which
allowed him to make prolonged microscopic observations of the growing cells for up to eight
months at a time. The Geissler chamber
was a section of glass tube that had been flattened
to provide a small growth zone or microcosm through which different media could be passed
(see [2, 17, 61]). These were probably the first flow chambers for the direct observation of
microorganisms, very similar to the pedoscops capillary system proposed almost a century
later [65], and analogous to current flow systems used in combination with CLSM (Confocal
laser scanning microscopy) (e.g. [95]). Winogradsky went on to use larger microcosms to
study sulfur bacteria, inoculating 3 5 litre flasks with pieces of muddy Butomus umbellatus
(flowering or grass rush) rhizome which were then maintained for up to 6 weeks whilst
measuring H2S production and sampling for bacteria. He also used small glass cylinders and
Petri dishes containing silica gels soaked with different nutrients to maintain and examine soil
cultures. Winogradsky used glass cylinders filled with sulfur spring water and sediments to
grow Beggiatoa. He subsequently used them to describe a variety of organisms, including
purple photosynthetic bacteria
. These cylinders, now known as Winogradsky’s columns,
containing various mixtures of sand, soil, sediments, and water, are ideally suited to
introducing microbiology students to selective culture methods, microbial community
succession, and stratification along chemical and physical gradients (e.g. [6, 74, 77, 78]).
Winogradsky’s columns are easily constructed and microbial changes are observable
within weeks (as an example, see Figures 2 and 3 showing the construction of a pair of
columns using river water and sediments collected from the banks of the Dnipro in Kyiv and
their development over a period of several weeks). A further refinement of the Winogradsky
column includes the use of agar to semi-solidify aqueous layers containing different nutrients
to allow the isolation of bacteria.
The ‘Geissler tube’ was the name given to early gas discharge tubes invented by the physicist Heinrich Geissler
in 1857. It is not clear how the Geissler chamber used by microbiologists might relate to this, though it was
used by Pasteur in the 1860’s [61].
Winogradsky was unaware at the time that they were capable of anaerobic anoxygenic photosynthesis [78].
From Winogradsky's Column to Contemporary Research ...
Figure 2. Constructing Winogradsky’s columns. We produced Winogradsky’s columns using river
water and sediments collected from the banks of the Dnipro. (Top left) The snow and ice-covered
Dnipro in Kyiv in January 2013. (Bottom left) Sediment samples diligently collected by two of the
Authors. (Right) Mariia Boretska (left) and Olena Moshynets (right) adding sediments and water to a
glass cylinder to produce the Winogradsky’s column. See Figure 3 for a series of images taken of these
columns over a period of several weeks. Images are from Mariia Boretska and Olena Moshynets.
Figure 3. Microbial development of the Winogradsky’s columns. The Dnipro river sediment and water
columns were maintained at room temperature (~20°C) for several weeks under anaerobic conditions
during which microbial colonisation and stratification occur. Shown here are a series of photographs of
two columns; the left-hand one was incubated in the dark, and the right-hand one exposed to natural
light. (Left to right) Immediately after construction; after 13, 23 and 37 days. Images are from Mariia
Boretska and Olena Moshynets.
In a study of the cycling of H2S and SO42- in Black Sea coastal waters, another early
pioneer of environmental microbiology, Mikhail Egunov, observed the development of a
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
bacterial film
between the upper SO42-rich layer and the lower H2Srich layer
, and
concluded that such a region occurred in the Black Sea at a depth of 200 m [18]; a sketch of
Egunov’s column was reproduced in a later publication by Winogradsky’s only student,
Vasily Omeliansky [57]). Although Winogradsky obtained his Beggiatoa from sulfur springs,
they have also been recovered from the Black Sea and other marine environments, including
deep-sea hydrothermal vent environments where they oxidize H2S to S0 and S0 to SO42-. In a
study of the mechanism and rate of sulfide oxidation in water, Yury Sorokin used an
experimental column to model the vertical gradient of the oxidation-reduction (redox)
potential of sea water and bacteria in the Black Sea in which he also cited Egunov’s earlier
work [87]. After a period of two weeks Sorokin observed the stratification of bacteria in two
principal regions of the column. The upper one contained H2Soxidizing Acidithiobacilli,
formerly known as Thiobacilli, between the upper O2rich water layer and the lower H2Srich
anaerobic water layer. The lower region was directly above a layer of agar at the bottom of
the column, containing sulfide and sulfates which diffused upwards into the water column. In
this lower region, sulfate-reduction was observed by the development of a ring of black
sulfate-reducing colonies on the cylinder walls. Sorokin’s column supported all of the
biological functions required for a complete sulfur cycle, despite being structurally a very
simple microcosm. Investigations of Black Sea microbiology continues today, almost 120
years after Egunov’s original work, and now includes modern approaches such as
metagenomics (reviewed by [96]). The ability to obtain bacterial sequences from
environmental samples, such as particulate matter or marine snow, can provide information
on bacterial diversity and community function. Sequences obtained from snow samples
recovered from the suboxic zone of the Black Sea suggest that the interior of these sinking
aggregates might support Mn (manganese) and SO42- reduction as well as H2S and S0
oxidation [25]. Although the redox gradient might be the most obvious large-scale chemical
gradient in the Black Sea, it is clear that there are smaller, local chemical gradients that might
also affect bacterial activity and help define micro-habitats. Micrometrescale gradients also
exist within sediment and soil pore networks and aggregates where they help define micro-
habitats and community function.
It is humbling to summarize the achievements of the founder of contemporary ecological
microbiology, albeit in such a superficial manner and without direct reference to his scientific
publications, but it is also a useful exercise to show the early development and use of
microcosms in microbiology. A microcosm can be simply defined as ‘a community, place, or
situation regarded as encapsulating in miniature the characteristics of something much
larger[63]. They have been used by biologists from a variety of disciplines to study
physiology, processes, and interactions over a range of scales, from single cell-traps to
laboratory cultures, aquaria, field trials, to the management of national parks, entire river
systems, lakes, coastal regions, and islands.
A ‘bacterial plate’ may be a more accurate translation of ‘бактериальная пластинка’ [18].
This is similar to Winogradsky’s observations of Beggiatoa behaviour under a cover-slip.
From Winogradsky's Column to Contemporary Research ...
Figure 4. Microcosms have been used to investigate aspects of microbiological ecology, adaptation and
evolution. (Top left) Plant-specific induction of Pf. SBW25 gene expression has been examined using
sugar beet seedlings grown in a vermiculite compost mixture (the plastic tube has a 1 cm diameter).
(Top middle) Bacterial evolution and biofilm-formation have been investigated using glass vials
containing liquid growth media (the glass vial has a 2.75 cm diameter). The microcosm on the left
contains wild-type Pf. SBW25 that grows in the liquid column, and in the microcosm on the right, the
evolved Wrinkly Spreader mutant forms a biofilm at the air-liquid interface. (Top right) The impact of
bacterial surfactant expression on water distribution within soil pore networks has been investigated
using sterile repacked soil aggregate cores (the core ring has a 37 mm diameter). (Bottom) Soil pore
structures visualized by X-ray CT have been printed out at a larger scale in plastic to allow the
investigation of fungal and bacterial colonization processes (the smallest cube is 1.8 cm wide). Images
are from Andrew Spiers and Ruth Falconer.
Although the use of microcosms in research has important caveats, most often associated
with the limited complexity of the system or the application of results to large-scale systems,
they nonetheless provide a very powerful experimental system for biological research. As an
example of this, we take the opportunity here to briefly present the research that colleagues
and ourselves have undertaken in this field over the last 15 years, utilizing artificial growth
media in glass vials and petri dishes, plants and repacked soil aggregate cores, and most
recently, plastic and transparent soils, to investigate aspects of microbial ecology, adaptation
and evolution (see Table 1 and Figure 4). Much of this work has involved the use of the soil
and plant-associated fluorescent pseudomonad, Pseudomonas fluorescens SBW25 (referred to
henceforth as Pf. SBW25), as a model organism in experimental evolution and biofilm studies
(e.g. [44, 72, 92, 93]) and plant-microbe interactions (e.g. [26, 27, 69, 83]). Pf. SBW25 was
isolated from a leaf of a sugar beet crop grown at the University Farm at Wytham, Oxford,
UK (SBW stands for Sugar Beet Wytham) [71]. Like other fluorescent pseudomonads, Pf.
SBW25 is a benign, plant growth-promoting bacterium, colonizing the leaves and roots of a
wide variety of crop plants and weeds. In general, P. fluorescens strains are not considered
plant pathogens [64], though like some other plant growth-promoting bacteria, Pf. SBW25
carries a defective pathogen-like type III secretion system [66].
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
Table 1. Contemporary investigations
Sampling and
Potential variables
and biofilmsb.
Glass vials
containing liquid
growth media.
Destructive sampling to
determine diversity,
fitness, biofilm growth,
strength and attachment.
Nutrients, O2, physical
disturbance, liquid
viscosity, interspecific
completion, phage and
plasmid co-evolution.
induction of bacterial
gene expressionc.
Sugar beet
seedlings grown in
vermiculite or
potting mixture.
Destructive recovery of
bacteria and sequencing;
re-testing to determine
expression levels and
fitness; Raman
Plant, bacteria and soil
type, temperature, water
and humidity.
Fitness advantage of
cellulose expression.d
mushroom caps.
Destructive recovery of
bacteria to determine
Mushroom and bacteria
type, presence of
predators and
Impact of bacterial
surfactants on soil
Repacked soil
aggregate cores.
Destructive determination
of water content.
Bacteria type and
distribution, soil type,
porosity, water content.
Fungal invasion of
soil pore networks.f
Printed plastic
model of real soil
photography and X-ray
CT imaging.
Structure (porosity, pore
dimensions, connectivity,
Transparent soil
Growth of plant
roots in Nafion
Non-destructive optical
and X-ray CT imaging;
root trajectories and
contact points, bacterial
distribution and activity.
Plant and bacteria type,
structure (porosity, pore
dimensions, connectivity,
etc.), Nafion surface
chemistry, pore liquid
chemistry and nutrients.
Investigations led by: (a), Angus Buckling, Michael Brockhurst, Rees Kassen and Paul Rainey; (b),
Paul Rainey and Andrew Spiers; (c), (d) and (e), Andrew Spiers; (f) and (g), Ruth Falconer and
Wilfred Otten.
Furthermore, many P. fluorescens strains including Pf. SBW25
produce soft rot-like
symptoms in plant tissues following physical damage, suggesting opportunistic pathogen-like
tendencies and a possible pathogenic origin for the species. Furthermore, the ability to
colonize new environments is a characteristic associated with the pseudomonads, and may be
enabled by relatively large genomes that include many sensory-regulatory elements [84, 90].
The Pf. SBW25 genome has been fully sequenced [83], and it can be manipulated easily
using standard molecular biology techniques.
Finally, the close similarity with the opportunistic human pathogen P. aeruginosa, the
plant pathogen P. syringae, and the archetypal soil isolate P. putida, make Pf. SBW25 an
ideal model organism for experimental studies.
Andrew Spiers, unpublished observations.
From Winogradsky's Column to Contemporary Research ...
Plant Microcosms
The ability of Pf. SBW25 to successfully colonise leaf and root surfaces depends on the
close adaptation of the bacteria to the local micro-scale chemical and physical conditions
prevailing on the plant, referred to as the phyllosphere, and in the narrow layer of soil in close
association with the roots, referred to as the rhizosphere. The ecological performance and
fitness of Pf. SBW25 in these different and dynamic environments is a complex phenotype, in
part explained by the ability of the bacteria to adjust gene expression patterns, enzyme
activities, physiology, metabolism, and behaviour in response to environmental opportunities
and signals. Various techniques have been developed to examine plant-specific induction of
bacterial gene expression, and one of these, known as IVET (in vivo expression technology),
was used by Paul Rainey’s group
to investigate Pf. SBW25 gene expression on sugar beet
seedlings in simple microcosms (see Figure 4) [26, 27, 69, 83]. In this work, the microcosm
consisted of small plastic tubes containing vermiculite or vermiculite-compost mixtures in
which individual sugar beet seeds were germinated and grown for 2 4 weeks. This period of
growth provided a selective environment in which only those IVET constructs responding to
specific, but unknown, plant signals survived.
The key to the IVET strategy was the use of Pf. SBW25 mutant strains that had been
deleted for an essential gene, either dapB needed to produce diaminopimelate required for the
cell wall, or panB required to produce the vitamin pantothenate, and a library of IVET
plasmids each containing a random fragment of the Pf. SBW25 genome inserted immediately
up-stream of a second copy of the deleted gene [26, 69]. If the genome fragment contained a
promoter sequence in the correct orientation, the plasmid-born copy of the essential gene
might be expressed allowing the host bacterium to survive
. Libraries of bacteria were used
to inoculate sugar beet seeds, and bacteria were recovered from the leaves, roots and bulk soil
after 2 4 weeks. These bacteria were tested on agar plates to exclude those plasmids
containing constitutively-active promoter sequences which had no environmental relevance.
The genome fragment in each IVET plasmid responding to specific environmental signals
was mapped back to the Pf. SBW25 genome by sequence analysis to determine which genes
were being expressed. Further experiments were conducted to assess the levels of gene
expression and their likely function in the sugar beet environment. Examples of genes
identified in this manner are those involved in nutrient acquisition, stress responses, and the
expression of antibiotics, cellulose and phytohormones [26, 27, 69, 83].
Unrelated research by the Rainey group investigating the Pf. SBW25 mutant Wrinkly
Spreader (WS) biofilm had just resulted in the identification of the cellulose biosynthesis
operon (wss) [92] and a demonstration that Pf. SBW25 could expressed partially-acetylated
cellulose in an air-liquid interface biofilm [92, 93] (discussed further in the following section;
see also the review by Spiers et al. [91]). Both biofilm-formation and the involvement of
cellulose in these structures is common amongst environmental Pseudomonas spp., giving
rise to the suggestion that the function or ecological role of biofilms in natural environments
might be to localize bacteria at the air-liquid interface in small bodies of water or liquid such
as those found in soil pores, on plant surfaces, or in plant tissues [73, 89, 91, 101]. Fitness
Department of Plant Sciences, University of Oxford, UK.
The genome fragment might contain i) no promoter activity; ii) a promoter only active in the plant or soil
environment; or iii) a promoter constitutively active in all environments including agar plates.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
assays had shown that there was an advantage to cellulose-expression by wild-type Pf.
SBW25 on sugar beet leaves and roots, but not in bulk soil [26], though this type of assay
does not provide a mechanistic explanation of how cellulose might improve fitness in these
environments. Recently, however, investigation of cellulose-expression in the related
pseudomonad, P. putida mt-2, suggest that it may provide a fitness advantage in water-
limiting conditions [53]. Examination of the survival and fitness advantage of a set of Brown
blotch-causing pseudomonads isolated from mushrooms, plus Pf. SBW25 and P. putida
KT2440 (a derivative of Pp. mt-2), support the view that cellulose might provide an
advantage by retaining water in micro-colonies and the absorption of water directly from
vapour under conditions of water stress, rather than by the formation of large biofilm
structures [44, 91]. The response of Pf. SBW25 to plant surfaces has also been examined by
single-cell Raman micro-spectroscopy [31]. This technique provides spectra reflecting the
whole-cell chemical composition of bacterial cells, and provides a means of monitoring
growth and metabolic status. Raman analysis is normally applied in the identification of
bacteria (e.g. [46]), but it might be used in a different manner to determine changes in
metabolism and physiology for bacteria growing on different nutrients or environments [33]).
Pf. SBW25 cells grown in vitro in defined minimal media containing single nutrient sources,
such as simple sugars, amino acids and TCA (tricarboxylic acid) cycle intermediates, were
found to show significantly different Raman spectral ‘fingerprints’, suggesting that the
technique could be used to differentiate bacterial cells growing in natural conditions on the
basis of local micro-scale chemical and physical conditions, growth history and strategy. For
example, this type of Raman analysis could differentiate between cells growing on fructose or
glucose (or glycerol), arginine or aspartic acid (or asparagine), etc., and could be used to
follow cells starving over a period of nine days [31], cells growing in high or lowO2
conditions or in a biofilm, and cells carrying the environmental Pseudomonas-associated
plasmid pQBR103 [36, 102].
Raman analysis could also differentiate between Pf. SBW25 cells growing in the sugar
beet rhizosphere and bulk soil without plants, suggesting that Pf. SBW25 responds to
different environmental signals which result in significant shifts in physiology and
Previously, it had been suggested that a correspondence between Raman spectra obtained
for bacteria growing in a natural environment and the same strain on defined growth media in
vitro might be used to infer the metabolic status of the former [33]. Pf. SBW25 recovered
from sugar beet leaves were found to have Raman spectral profiles more similar to Pf.
SBW25 grown in vitro on fructose or aspartic acid, rather than on glucose or arginine, and
where quite dissimilar to starved cells [31]. This suggests that Pf. SBW25 growth on the
leaves of sugar beet is likely to be neither carbon-catabolite-repressed nor carbon-limited.
Wei Huang et al.
have also demonstrated the utility of Raman analysis further by showing
that it can be combined with both SIP (stable isotope probing) and FISH (fluorescence in situ
hybridization) [32, 35]. Raman can also be combined with laser optical tweezers to trap and
recover individual bacteria from solution [34, 107].
Centre for Ecology and Hydrology, Oxford, UK.
From Winogradsky's Column to Contemporary Research ...
Experimental Evolution and Biofilms
Experimental evolution has been used by ecologists in an attempt to understand the
dynamics of diversifying and adapting populations, and more recently, in an attempt to link
evolutionary dynamics, fitness, and niche specialisation, with an understanding of the
mechanistic link between the underlying organismal molecular biology and environmental
factors (for reviews, see [10, 41, 70]). Pf. SBW25 has been used as a model organism in
experimental evolution studies using simple glass microcosms and was first described by Paul
Rainey and Michael Travisano [72] (see Figure 4; see also Chapter 5 and references therein).
When wild-type Pf. SBW25 is inoculated into nutrient-rich King’s B liquid medium [43] the
population will grow over a period of several days and accumulate mutants. One of the most
visually obvious of these mutants is the Wrinkly Spreader, characterized by a wrinkled colony
morphology on King’s B agar plates. The Wrinkly Spreader also differs from the ancestral,
wild-type Pf. SBW25 in that it no longer colonizes the liquid column of static King’s B
microcosms, but grows at the air-liquid interface by forming a robust biofilm (for a
description of the evolution of the Wrinkly Spreader, see [29]). Furthermore, in static
microcosms, the Wrinkly Spreader has a fitness advantage over the ancestral Pf. SBW25 and
other non-biofilmforming competitors [29, 92]. This advantage demonstrates that the
Wrinkly Spreader is not simply one of many different random mutants that might arise during
the diversification of Pf. SBW25 populations, but is an evolved genotype, representing a
fitness or adaptive leap from the ancestral Pf. SBW25 and having a new niche preference for
the air-liquid interface formerly not colonized by Pf. SBW25. In shaken microcosms where
biofilms cannot form, the Wrinkly Spreader does not have a fitness advantage over wild-type
Pf. SBW25 or other non-biofilmforming strains [29, 92], and on agar plates, the Wrinkly
Spreader is at a disadvantage and is genetically unstable [88]. Investigation of the distribution
of O2 in static microcosms by Andrew Spiers’ group
has shown that the Pf. SBW25
colonisers modify the environment by creating an O2 gradient within hours of introduction
into static King’s B microcosms [45]. The O2 gradient divides the microcosm into an upper,
O2-rich zone of ~200 µm, and a lower O2-depleted zone. These zones present the developing,
diversifying Pf. SBW25 population with new niches to occupy. Although Wrinkly Spreaders
may appear anywhere in the liquid column, they are more likely to appear in the O2-rich zone
as this supports faster growth; once they appear and localize to the air-liquid interface, they
begin to produce a biofilm which will intercept O2 diffusing into the liquid column from the
air above. By intercepting this O2, Wrinkly Spreader cells at the air-liquid interface will grow
faster than the ancestral wild-type Pf. SBW25 and other non-biofilmforming mutants lower
down in the liquid column, thus explaining the fitness advantage of the Wrinkly Spreader
[45]. As the Wrinkly Spreader biofilm develops, there is opportunity for further evolution as
the O2 gradient now subdivides the biofilm itself into an O2-rich surface layer of ~100 µm
and a lower O2-depleted zone.
In a similar air-liquid interface biofilm produced by Gluconacetobacter xylinus (formerly
known as Acetobacter xylinum), the opposing O2 and nutrient gradients combine to define an
optimal growth zone 50 100 µm deep, where there is sufficient O2 diffusing down from the
air above and nutrients diffusing upwards from the liquid column [103]. Recent experiments
by the Spiers’ group suggest that the fitness advantage of the Wrinkly Spreader in static
The SIMBIOS Centre, University of Abertay Dundee, UK.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
King’s B microcosms also depends on the relative availability of O2 and nutrients, as well as
on the relative expense of biofilm-formation versus the cost of maintaining position at the air-
liquid interface by swimming motility. Growth in O2-rich conditions, and in a biofilm rather
than as individual free-swimming planktonic cells, has a big impact on the metabolic and
physiological status of bacteria, as shown by single cell Raman analysis of Pf. SBW25 [36]
and a similar analysis of a different P. fluorescens strain investigated by ATRFTIR
(attenuated total reflection Fourier transform infrared spectroscopy) [67]. The relative ease
in distinguishing Wrinkly Spreader colonies from wild-type Pf. SBW25 and other mutant
classes on King’s B agar plates has meant that studying diversification and adaptation is
relatively simple
. For example, the impact on physical disturbance, nutritional complexity,
and the presence of bacteriophage
have all been examined using this simple microcosm
system [8, 9, 40, 62]. The molecular biology underlying the WS (Wrinkly Spreader)
phenotype, including the colony morphology and biofilm, has also been examined in detail [7,
92, 93, 94]. Using a mini-transposon mutagenesis approach, a number of mini-Tn5 mutants of
the archetypal Wrinkly Spreader producing wild-type Pf. SBW25like colonies and unable to
form biofilms, were isolated and the position of the mini-transposon located on the genome
determined by sequencing. This approach and subsequent testing determined that the WS
phenotype required the expression of partially-acetylated cellulose and an unidentified pili-
like attachment factor [92, 93, 94], and that it was activated by a single amino residue change
in a regulatory sub-unit of a membrane-associated chemoreceptor [7]. This mutation activated
a diguanylate cyclase known as WspR, to produce cyclic-di-GMP (cyclic di-3’,5’-guanylate)
which then activated the cellulose synthase complex and the expression of the attachment
factor [7, 48] (for a more detailed description of the WS phenotype see [29]; for a review of
cellulose-expression and biofilm-formation amongst the pseudomonads, see [91]; and for a
review of cyclic-di-GMP signalling in bacteria, see [38]).
SIMBIOS and Soil Research
The Scottish Informatics, Mathematics, Biology and Statistics (SIMBIOS) Centre was
established at the University of Abertay Dundee to support a range of interdisciplinary
research activities, including investigations of the micro-scale structure of soil and the use of
modelling approaches, to understand how spatial heterogeneity at the microscopic scale
affects biophysical behaviour at larger scales. Key to this work has been the use of high
resolution X-ray CT (micro-X-ray computed tomography) to non-destructively visualize and
quantify soil pore networks, resolving features down to 3 µm resolution and differentiating
between materials of the basis of electron density (reviewed by [97]). A 2-D (two-
dimensional) image of the physical structure of a soil aggregate, and 3-D (three-dimensional)
images of a soil sample visualized by X-ray CT are provided in Figure 5 (images such as
these are usually manipulated on screens where they can be rotated in 3-D space allowing the
viewer an excellent impression of pore spaces and networks). SIMBIOS uses X-ray CT to
investigate micro-habitat structures in natural soil and repacked soil aggregates, and are
Experimental evolution is studied using other bacteria which do not produce Wrinkly Spreader-like mutants. In
these systems, more laborious or expensive procedures must be used to distinguish between mutants.
A bacterial virus.
From Winogradsky's Column to Contemporary Research ...
developing in parallel new statistical and image analysis tools required for this type of work
[54, 56]. They are also combining the 3-D microtomographs of soil structure with other
techniques, such as aggregate sampling and thin sectioning used to investigate bacterial
colonization (e.g. [55, 56]) and SEMEDX (scanning electron microscopy energy-
dispersive X-ray spectroscopy) to map elemental distributions [30, 58]. Natural soil structures
are hard if not impossible to replicate for research purposes (e.g. example, to investigate
aspects of bacterial colonization, community structure and function, plant root development,
and plant-bacterial interactions).
Soil microcosms are generally constructed by re-packing sieved soil aggregates (see
Figure 4), and X-ray CT can be used to quantify pore structure in terms of porosity, pore
dimensions and connectivity, etc., of these artificial environments. However, X-ray CT is
limited by current ability to differentiate between materials of similar electron density (e.g.
differentiating the air, water, organic and inorganic matter can be problematic). Under some
circumstances, X-ray CT may be able to visualize water films in soil pore networks (e.g.
[99]), and in the near future, it may be possible to map the spatial distribution of water within
the soil pore network at the micrometre scale.
Figure 5. X-ray CT can be used to visualize the physical structure of soils. Grey-scale images are used
to indicate different densities ranging from air-filled pore spaces (white) to dense aggregates (grey) and
solid particles (black). (Left) A 2-D slice showing the internal structure of a soil aggregate (2 x 2 mm)
at a spatial resolution of 3 µm. (Right) A 3-D projection of a soil sample (1.4 x 1.4 x 2.7 cm) is shown
as a grey-scale solid and again showing only the pore spaces, at a spatial resolution of 32 µm. Images
are from Wilfred Otten.
Plant roots in soil can also be visualised by X-ray CT [100], and Sonja Schmidt et al.
have used it to investigate the interaction between roots and soil particles at the micrometre
scale, finding that the level of root-soil contact increased with decreasing particle size, and
possibly also as a result of changes in particle packing around the root [80].
The SIMBIOS Centre, University of Abertay Dundee, UK, and the James Hutton Institute, Invergowrie, UK.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
Soil Microorganisms
The glass slides used by Cholodny [12, 13] and other early investigators to visualize soil
microbiology whilst retaining some spatial detail are largely forgotten today as focus has
shifted to the use of molecular techniques to investigate community function and diversity.
However, there is growing recognition that there is a need to investigate microbial community
function at the micrometre in the soil pore networks, where local physical and chemical
conditions, and inter-species interactions are key to community function. The glass slides
used by Cholodny are often viewed as problematic, insofar the slide represents a large, solid,
and impermeable surface that alters soil structure and might restrict plant root and fungal
development, and microbial mobility. However, these problems are reduced by using smaller
sampling devices with different surface chemistries and permeabilities. Plastic films such as
PET (polyethylene terephtalate) can be used instead of glass slides, and are readily cut into
smaller samplers of 2 5 mm2.
Olena Moshynets et al.
have shown that both Pf. SBW25 and P. putida KT2440 readily
attach and detach from PET films in vitro, and pre-conditioning with soil wash suspensions
can be used to modify the PET surface to increase bacterial attachment [50]. PET films buried
in soil microcosms are rapidly colonized by microorganisms which could be investigated by
CLSM or recovered onto agar plates; examples of this are shown in Figure 6 (like the soil
landscapes produced by Winogradsky, these images are visually more exciting than the X-ray
images and give a good impression of the microbiological complexity of soils at the
micrometre scale). PET film samplers buried in soil microcosms can be non-destructively
imaged by X-ray CT to map soil aggregatefilm contact points and pore spaces [50]. In this
manner, it may be possible to link specific micro-habitats identified by X-ray CT with
molecular analyses conducted on the recovered film still retaining spatial detail (e.g. FISH or
PCR (polymerase chain reaction) approaches to identify species and to examine gene
expression patterns). However, some techniques such as Raman may not be readily applicable
due to the auto-fluorescence spectrum of the PET polymer
. The PET film samplers have
also been used to investigate bacterial colonization of bamboo tissues and the rhizosphere of
Brassica napus (rapeseed) [51, 52]. Archana Juyal and Thilo Eickhorst
are investigating the
dynamics of bacterial colonization of soil pore networks. They are using repacked soil
aggregate microcosms inoculated with model microorganisms such as Bacillus subtilis and
Pf. SBW25. These microcosms are embedded in resin and thin sections prepared for
Two examples of the images obtained using this technique are provided in Figure 7; in
the first, all microorganisms are stained using a DNA-binding fluorescent dye, and in the
second, Betaproteobacteria cells are specifically visualized by FISH (both images are of the
same region of the thin section). Although the embedding process can result in sample
shrinkage, they are able to retrospectively identify the position of sliced sections in X-ray CT
images of the original core, similar to the mapping of buried PET film samplers. Thilo
Eickhorst is also developing CARD-FISH (catalyzed reporter depositionFISH) for use with
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine, and
during visits to the SIMBIOS Centre, University of Abertay Dundee, UK.
Wei Huang, unpublished observations, personal communication to Andrew Spiers.
The SIMBIOS Centre, University of Abertay Dundee, UK, and the Institute of Soil Science, University of
Bremen, Germany, respectively.
From Winogradsky's Column to Contemporary Research ...
the thin sections, as this technique promises to be a more sensitive method than standard
FISH or the use of fluorescent stains that bind to DNA (deoxyribonucleic acid) to detect the
presence of microorganisms [5, 19, 81].
Figure 6. PET film samplers can be used to visualize the microbiological complexity of soils. Films
were buried in soil for two weeks before recovery and staining with the DNA-binding fluorescent dye,
Acridine orange (3,6-acridinediamine), to visualize microorganisms. Sections of film surfaces (60 x 60
µm) were inspected by CLSM (Confocal laser scanning microscopy), and in these images, many
different types of microorganism can be distinguished but not identified. Images from Olena
Figure 7. Bacteria can be identified in pore spaces in thin sections of soil embedded in resin.
Fluorescent microscopy was used to image a region of a thin section of resin-embedded paddy soil
(sandy loam) using two different techniques for the same field of view (100 x 150 µm). (Left) All
microorganisms are visualized using the DNA-binding fluorescent dye, DAPI (4',6-diamidino-2-
phenylindole). (Right) Betaproteobacteria are visualized by FISH using a specific 16S rRNA probe.
Images from Thilo Eickhorst.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
Although generally 16S rRNAtargeted oligonucleotide probes are used for FISH and
CARD-FISH to identify microorganisms at a number of different phylogenetic levels, it is
possible to use gene-specific probes that could also indicate whether individual bacteria are
expressing genes involved in biological processes at the micrometre scale within soil pores.
For example, the spatial clustering of bacterial nitrogen fixation genes within soil is probably
due to the separation of populations across aggregates of different size classes, caused by
differences in the temporal stability of aggregates as niches for microbial communities [37].
Although the identification of thin sections in the 3-D X-ray CT images of the original core
were done manually, more rigorous statistical methods have been developed by Simona
Hapca et al.
This work originated from collaboration with Clare Wilson
who was studying micro-
spatial heterogeneity of SOC (soil organic carbon), Fe (iron) and Al (aluminum) in soils using
SEM-EDX to produce two-dimensional maps which were then combined with the 3-D X-ray
CT images [58].
Impact of Bacterial Surfactants on Soil Hydrology
Pf. SBW25 and other soil and plant-associated pseudomonads are known to express
powerful surfactants such as the CLP (cyclic lipopeptide) viscosin, involved in a variety of
roles including motility, biofilm formation, anti-bacterial activity, the inhibition of protist and
nematode predation, lysis of fungi and oomycetes, and the induction of systemic resistance in
plants (reviewed by [15, 68]).
However, surfactants also have a simpler impact in water where they reduce the liquid
surface tension (γ) and alter the shape of liquid bodies, especially at the meniscus or the air-
liquid-solid interface. The impact of reduced γ on liquid surface geometry is significant and
can be illustrated by the drop collapse assay where liquids with high–γ (e.g. water) produce
uniform hemispherical drops on glass slides whilst low–γ solutions produce irregular
flattened shapes.
However, the ecological value to bacteria of directly modifying or manipulating water
geometry through the expression of surfactants had not been considered, despite the fact that
water distribution in fully and partially-saturated pores, pore-wall surface films and droplets
is a key factor in bacterial colonisation and activity in soils [14, 108]. Using repacked soil
aggregate microcosms (see Figure 4), the Spiers’ group demonstrated that the volumetric
water content of soil was significantly lowered by Pf. SBW25 expressing the viscosin
compared to a surfactant-deficient mutant [21]. Furthermore, six of fifteen pseudomonads
isolated directly from soil shown to express surfactants in vitro were found to have a similar
impact on water content when compared to sterile microcosms [21]. These findings indicate
that surfactant-expressing pseudomonads could modify local soil-water distributions within
soil pore networks, and that surfactants may therefore play a significant hydrological role in
soils in addition to their recognised biological activities. As X-ray CT technology develops, it
may be possible to investigate bacterial surfactant-mediated changes to local soil water
distribution patterns at the micrometre scale in the future.
The SIMBIOS Centre, University of Abertay Dundee, UK.
School of Biological and Environmental Sciences, University of Stirling, UK.
From Winogradsky's Column to Contemporary Research ...
Plastic and Transparent Soils
As an extension of the experimental investigation and modelling of fungal invasion of
soil pore networks [60], Ruth Falconer and Wilfred Otten
have employed 3-D printing
techniques to produce realistic plastic soil structures using data derived from X-ray CT of
native soil cores [59] (see Figure 4).
This printing technology can produce multiple replicates of the same native structure, and
by transforming the data set, related structures differing in a key parameter such as total
porosity can be produced for testing.
Furthermore, the plastic provides a physically and chemically homogeneous surface,
allowing the effect of soil structure to be investigated without the complication of chemical
However, just like the PET film samplers, the chemical surface of the plastic soils might
be modified to allow subsequent investigations of physical and chemical heterogeneity in
microcosms in which structure remains constant.
In some circumstances, realistic soil structure may not be a priority where research is
focusing on plant root development or bacterial-root interactions. Helen Downie et al.
developed OPT (optical projection tomography) to image roots and bacteria in a novel
‘transparent soil’ microcosm [16]. These were constructed using Nafion particles (a
sulfonated tetrafluoroethylenebased fluoropolymer-copolymer) to provide a soil-like pore
network that could be partially saturated with a nutrient solution to allow the growth of
Latuca sativa (lettuce) and Nicotiana benthamiana (a close relative of tobacco frequently
used by plant scientists) seedlings.
Immediately before visualization, the pores were saturated with a RI (refractive index)-
matched solution, rendering the particles transparent and allowing the direct imaging of roots
and bacteria.
The pore structure itself can be visualized using fluorescent dyes that adsorb onto the
particles, while other dyes or fluorescent protein-labelling can be used to label roots or
bacteria [16].
Winogradsky was the father of microbial ecology, developing it as a separate discipline,
distinct from and as valid as medical microbiology led by the disciples of Koch. He
championed the need to study soil microorganisms in their natural habitat, and pioneered the
use of microcosms in which biological processes could be investigated. Winogradsky’s Direct
method has matured into a truly interdisciplinary research approach that is highly topical to
today’s environmental microbiologists and soil biophysicists. We use this approach today,
employing a variety of simple microcosm systems to examine aspects of microbial ecology,
and strongly encourage other microbiologists to do likewise in their own research.
The SIMBIOS Centre, University of Abertay Dundee, UK.
The SIMBIOS Centre, University of Abertay Dundee, UK, and the James Hutton Institute, Invergowrie, UK.
Olena Moshynets, Mariia Boretska and Andrew J. Spiers
Supplementary Table 1. The professional career of S.N. Winogradsky
1877 1881 Entered the Department of Natural Sciences of the Physico-Mathematical Faculty, University of St.
Petersburg. He then chose plant physiology as his major, working as an undergraduate research student
with the botanist-physiologist Andrey Famintsyn. Awarded a Diploma of Science in 1881 and continued
his research studies with Famintsyn.
1881 1884 First St Petersburg research period. Worked with Famintsyn on bacterial fermentation. Published his
first research work on the nutrition and growth physiology of the yeast Candida vini. Awarded the degree
of Master of Science in 1884.
1885 1888 Strasbourg research period. Joined the botanist and mycologist Anton DeBary at the University of
Strasbourg. He investigated the sulfur-oxidising bacterium Beggiatoa, proving the monomorphism of so-
called ‘high bacteria’. Discovered the autotrophic assimilation of CO2 by bacteria and formulated the
theory of chemolithotrophy. Developed selective (elective) conditions for bacteria cultivation.
1888 1891 Zurich research period. He developed his chemistry skills with Ernst Schultz and Otto Roth at the
Swiss Polytechnic Institute in Zurich, where he investigated sulfur and iron bacterium, and bacterial
nitrification. Isolated pure cultures of Nitrosomonas and Nitrobacter. Lectured at the Zurich Polytechnic
Institute. He rejected an offer to join the Pasteur Institute in 1891, preferring to return to Russia.
1891 1905 Second St Petersburg research period. Became the Head of the General Microbiology Department of
the Imperial State Institute of Experimental Medicine at St. Petersburg (resigned as Director in 1905 but
remained a member until 1912). Corresponding Member of the Academy of Sciences of France (1902).
In 1903 he and Omeliansky organized the Russian Microbiological Society, where he was a director for
two years. Participated in the development of anti-plague measures in Russia. In 1894 he proved the
existence of anaerobic nitrogen-fixing bacteria, isolated and described Clostridium pasteurianum.
1905 1920 Due to health problems he left St. Petersburg and returned to his estate in Gorodok, Ukraine. Honorary
Member of the German Botanical Society (1907). Foreign Member of the Royal Academy of Agriculture
of Sweden (1908). Corresponding Member of the Academy of Medicine of Torino (1909). Honorary
Member of the Russian Microbiological Society (1910). Corresponding Member of the Microbiological
Society of Holland (1911). Honorary Member of the Polytechnic Institute of Riga (1912). In 1916 he
moved to Odessa. Foreign member of the Royal Society in London (1918). In 1920 he left Odessa
following the Russian Revolution by French warship as a Corresponding Member of the French
Academy of Sciences.
1921 1922 Joined the Agricultural Institute of the University of Belgrade, Yugoslavia. Member of the Czechoslovak
Botanical Society and a Professor of the University of Belgrade (1921). In 1922 he accepted an invitation
from Emil Roux, director of the Pasteur Institute and moved to Paris.
1923 1953 Paris research period. Continued working on nitrogen-fixation in bacteria and soils; he was the first to
study soils as an environment. Developed his Direct method to investigate the biological, chemical and
physical characteristics of soil, beginning the modern disciplines of ecological microbiology and soil
microbiology. Head of the Laboratory of Agricultural Microbiology of the Pasteur Institute in Brie-
Comte-Robert (Paris). Honorary Member of the Academy of Sciences of the USSR (1923). Member of
the Institute of France as a Foreign member of the Academy of Science, Foreign Member of the English
Royal Society and a Honorary Member of the International Society of Soil Sciences (1924). Foreign
Member of the Academy of Sciences of France (1925). Corresponding Member of the American
Microbiological Society (1926). Honorary Member of the Biological Society of France (1929). Member
of the Scientific Society of Italy (1931). Member of the Royal Academy of Science in Amsterdam
(1932). Foreign Member of the Royal Academy of Science of Sweden (1934). Awarded the
Leeuwenhoek Medal (1935). Honorary President of the IV International Botanical Congress in
Amsterdam (1935). Honorary Member of the Society of Biological Chemists of India and the Society of
American Bacteriologists (1936). Honorary Vice-President of the II International Microbiological
Congress in London (1936). Awarded a Diploma of Ministry of New Jersey (1936). Honorary President
of the International Botanical Congress in Stockholm (1940) and the IV International Microbiological
Congress in Copenhagen (1947). Honorary Member of the Society of Soil Sciences of Florida (1950).
Honorary Member of the Society of General Microbiologists of the UK (1952).
1953 Died in Brie-Comte-Robert.
This is not a comprehensive list of Winogradsky’s research career or accomplishments.
From Winogradsky's Column to Contemporary Research ...
We thank Dmitry Musolin, St. Petersburg State Forest Technical University and St.
Petersburg State University, and Grozdilova Ludmila, Institute of Zoology of the Russian
Academy of Sciences, St. Petersburg, for their assistance in our search of the literature. We
also thank Dmitry Irodov and Oleksy Burlak for their assistance accessing some of the early
publications and photography of the columns, and members of SIMBIOS who provided
images and comments on the developing manuscript. Andrew Spiers is a member of SAGES
(Scottish Alliance for Geoscience Environment and Society), and is supported by the
SIMBIOS Centre and the University of Abertay Dundee. The University of Abertay Dundee
is a charity registered in Scotland, No: SC016040.
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... Permukaan kolom terkena oksigen, semakin rendah kolom, semakin sedikit oksigen yang tereduksi ke bawah, ini merupakan zona anaerobik. Permukaan dan tepi kolom terkena cahaya sehingga spektrum pertumbuhan mikroba dapat digambarkan, dari mikroba yang membutuhkan oksigen dan cahaya hingga mikroba yang membutuhkan cahaya tetapi oksigen tidak (Moshynets et al., 2013;Esteban et al., 2015;Babcsányi et al., 2017). Sumber karbon atau sulfur yang ditambahkan dapat mendorong pertumbuhan mikroba di kolom Winogradsky. ...
Conference Paper
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Bakteri fotosintetik merupakan mikroba yang mampu melakukan fotosintesis anoksigenik. Bakteri ini mampu menggunakan sumber karbon proses fotosintesis. Salah satu metode yang dapat digunakan untuk mengetahui adanya bakteri fotosintetik adalah kolom Winogradsky. Kolom Winogradsky adalah kultur pengayaan yang biasanya dibuat dengan mengisi silinder transparan dengan tanah atau sedimen dan diinkubasi dalam cahaya. Tujuan penelitian ini adalah untuk mengetahui mengetahui bakteri fotosintetik yang berpotensi muncul terdapat di dalam tanah lumpur dengan menggunakan kolom Winogradsky. Metode penelitian yang digunakan adalah deskriptif kualitatif dan kolom Winogradsky. Hasil penelitian menunjukkan bahwa sumber karbon dengan tepung terigu terdapat warna merah pada air dan terbentuk lapisan berwarna merah pada permukaan air. Kemudian, sumber sulfur dengan kuning telur terdapat warna cokelat pada air dengan lapisan berwarna cokelat tua pada permukaan air dan lapisan permukaan tanah berwarna cokelat gelap. Warna merah dan cokelat menunjukkan potensi muncul bakteri fotosintetik dari tanah lumpur.
... For the biofilm inactivation assay, LB cultures were diluted to 10% and 200 µL aliquots transferred to the wells of a 96-well plate that was incubated at 37 • C for 24 h before the biocide inactivation assay. For CLSM and the eDNA yield assay, 10% dilutions were used to inoculate 30 mL glass vials (microcosms) containing 2-5 mL LB that were incubated statically at 37 • C for 3-5 days to produce biofilms [80]. The biofilms were air-dried at room temperature for 14 days before eDNA yield analysis. ...
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The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.
... En cuanto a los gradientes de NO 2 y NH 4 , presentan un comportamiento normal, al ser desechos de los microorganismos (Figuras 4D, 4F, 5D y 5F) (Leiva et al., 2017). Los resultados obtenidos fueron similares a los obtenidos en el río Hoosie en Estados Unidos (Rundell et al., 2014), y en el río Dniéper en Ucrania (Moshynets et al., 2013). ...
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El ciclo de azufre es uno de los ciclos biogeoquímicos más importantes del planeta. Sin embargo, la contaminación generada por las actividades antrópicas puede modificar este ciclo, sobre todo en los ecosistemas acuáticos, al ser más vulnerables. El principal objetivo del estudio fue crear columnas de Winogradsky a partir de agua y sedimentos de cuatro ecosistemas acuáticos en la Región Amazonas, dos lénticos y dos lóticos, estudiando los microorganismos relacionados con el ciclo del azufre que se desarrolla en cada ecosistema, y cómo están afectados. Para ello, se determinaron los gradientes fisicoquímicos generados y cómo se agrupan, tanto estos, como los ecosistemas y las columnas creadas. Se analizaron en cada ecosistema acuático y en cada columna de Winogradsky, parámetros fisicoquímicos como oxígeno disuelto (OD) sulfatos (SO4), fosfatos (PO4), nitratos (NO3), nitritos (NO2) y amonio (NH4). Las columnas de Winogradsky creadas no mostraron todos los estratos que deberían presentar. Los gradientes fisicoquímicos fueron, en general, normales, excepto el del OD en los ecosistemas lénticos, indicando una posible eutrofización. Se observó que los ecosistemas acuáticos y las columnas de Winogradsky creadas forman tres grupos muy marcados, en uno se agrupan las columnas de los ecosistemas lénticos, en otro las columnas de los lóticos y en otro todos los ecosistemas. Los parámetros de SO4 y PO4 forman un grupo totalmente separado del resto de variables en cuanto a la agrupación de los parámetros fisicoquímicos. Por tanto, el ciclo del azufre en los ecosistemas acuáticos estudiados de la Región Amazonas se vio afectado, principalmente, por actividades antrópicas como la agricultura y la ganadería.
... We have been investigating this by comparing biofilms produced by environmental Pseudomonas spp. isolates, using relatively large static microcosms in 30-ml glass universal vials containing liquid growth medium [44,47,48]. These allow us to undertake combined biofilm assays which determine growth, attachment to the vial walls, and biofilm strength [49][50][51]. ...
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At nuclear hazard sites, such as the Chernobyl reactor sarcophagus or Fukushima Nuclear Power Plant, radiation is one of the main factors influencing microbial communities including those involved in microbially influenced corrosion (MIC) of metal structures. By studying the impact of radiation on corrosion-relevant bacteria it may be possible in the future to predict changes in MIC. We believe that the composition and function of natural multi-species biofilms will change when exposed to the stress of ionizing radiation. To address this possibility, biofilm formation by Pseudomonas pseudoalcaligenes and Stenotrophomonas maltophilia were studied after exposure to a range of radiation dosages. Altered planktonic cell morphologies and biofilm architectures on submerged glass surfaces were noted 3 – 7 days after low-doasage sub-lethal irradiation (5.3 Gy) of samples at the micro-colony, macro-colony and mature biofilm stages of development. Furthermore, significant differences in the percentage area covered by biofilms and the release of viable planktonic cells was also noted. These observations suggested that exposure, considered as insignificant levels of irradiation, can be enough to alter biofilm formation of corrosion-relevant bacteria. Such low dosage radiation may have significant impact on soil microbial communities in nuclear hazard sites, potentially altering the MIC of exposed metal structures, their stability and service life of underground metal constructions.
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The Winogradsky column is used as a microcosm to mimic both the microbial diversity and the ecological relationships between the organisms in lake sediments. In this study, a pyrosequencing approach was used to obtain a more complete list of the microbial organisms present in such columns and their ratios in different layers of this microcosm. Overall, 27 different phyla in these columns were detected in these columns, most (20 phyla) belonged to bacteria. Based on this study, Proteobacteria (mostly Sphingomonadales), Cyanobacteria (mostly Oscillatoriales) and Bacteroidetes (mostly Flavobacteriales) were the dominant microorganisms in the water, middle, and bottom layers of this column, respectively. Although the majority of organism in the water layer were photoautotrophic organisms, the ratio of the phototrophic organisms decreased in the lower layers, replaced by chemoheterotrophic bacteria. Furthermore, the proportion of aerobic chemoheterotrophic bacteria was greater in the higher layers of the column in comparison to the bottom. The green and purple sulfur phototrophic bacteria inhabited the bottom and middle of these columns, with none of them found in the water layer. Although the sulfur oxidizing bacteria were the dominant chemolithotrophic bacteria in the water layer, their ratio decreases in lower layers, being replaced with nitrogen oxidizing bacteria in the middle and bottom layers. Overall, the microbial population of these layers changes from a phototrophic and aerobic chemoheterotrophic organisms in the water layer to a mostly anaerobic chemoheterotrophic population of bacteria in the bottom layers.
Understanding evolution is crucial to modern biology, but most teachers would assume that practical demonstrations of evolution in school laboratories are unfeasible. However, perhaps they have not heard of 'evolution in a test tube' and how Wrinkly Spreaders can form the basis for both practical demonstrations of bacterial evolution and further work.
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Biological soil thin-sections and a combination of image analysis and geostatistical tools were used to conduct a detailed investigation into the distribution of bacteria in soil and their relationship with pores. The presence of spatial patterns in the distribution of bacteria was demonstrated at the microscale, with ranges of spatial autocorrelation of 1 mm and below. Bacterial density gradients were found within bacterial patches in topsoil samples and also in one subsoil sample. Bacterial density patches displayed a mosaic of high and low values in the remaining subsoil samples. Anisotropy was detected in the spatial structure of pores, but was not detected in relation to the distribution of bacteria. No marked trend as a function of distance to the nearest pore was observed in bacterial density values in the topsoil, but in the subsoil bacterial density was greatest close to pores and decreased thereafter. Bacterial aggregation was greatest in the cropped topsoil, though no consistent trends were found in the degree of bacterial aggregation as a function of distance to the nearest pore. The implications of the results presented for modelling and predicting bacterial activity in soil are discussed.
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(From the Introduction) Bacterial cellulose was first isolated from the air-liquid (A-L) interface biofilm produced by Bacterium xylinum in 1886, an acetic acid bacterium strain which would probably now be recognised as Gluconacetobacter xylinus (formerly Acetobacter xylinum) or a related species. Over the following century, more acetic acid bacteria and additional Proteobacter were found to produce cellulose. Cellulose-producing bacteria include a mixture of gut commensals, plant and animal pathogens, and all share soil as a common secondary habitat. It is likely that cellulose provides protection against physical disturbance, predation or other environmental stresses common to these diverse environments. The biochemistry of bacterial cellulose expression has been studied extensively for Gluconacetobacter, and this understanding has been used as a model for enteric bacteria and pseudomonads. Experimental reports of bacteria expressing cellulose are increasing, as well as the annotation of putative cellulose synthase-like operons in bacterial whole-genome sequences, suggesting that an increasingly wider range of bacteria may be capable of producing cellulose. Our interest in bacterial cellulose began with the experimental evolution of the soil and plant-associated pseudomonad, Pseudomonas fluorescens SBW25. This resulted in a novel biofilm–forming adaptive mutant known as the Wrinkly Spreader (WS). Subsequent investigation of the WS phenotype identified partially-acetylated cellulose as the main matrix component of the biofilm. The pseudomonads are a highly diverse genus, and biofilm-formation and cellulose expression are now known to be common amongst the water, soil, plant-associated and plant-pathogenic environmental pseudomonads. However, the ecological role of cellulose and the fitness advantage it confers to these bacteria is poorly understood. Here we provide a review of our work focussing on biofilm-formation and cellulose expression by SBW25 and other environmental pseudomonads. (A PDF is available on request)
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The identification of cultivated and uncultivated microorganisms via microbiological and molecular tools is a traditional way to study plant associated micro-communities. However, the functional activity of microorganisms in a micro-community associated with a plant is comprised of a multitude of complex cellular interactions in a specific niche. Therefore, it is not enough to know the composition of a microbial community if one wishes to understand the cytoecology that grows out of all these fine interactions. Even worse – in spite of the significant amount of data regarding the importance of endophytic micro-organisms for plants, little progress has been made with respect to the spatial architecture of the microbial communities and their interactions. Here we offer a new methodological approach to allow the combination of structural, microbiological and molecular techniques for direct observation of plant-microbial interactions under environmental conditions. In order to study plant-microbe relationships, a few effective modifications of the decades old methods of Cholodny were put forward, and these have been used to visualize the bacterial and fungal endoflora of the bamboo Phyllostachys atrovaginata. Different cell forms and community architectures have been observed along the culm. The method used here offers a lot of potential to complement the metagenomic approach to bacterial ecology which dominates the field today.
Prokaryotes are well recognized as essential members of the biosphere. They inhabit all possible locations in which life exists from those offering ideal conditions for growth and reproduction to those representing extreme environments at the borderline of abiotic conditions. © 2013 Springer-Verlag Berlin Heidelberg. All rights are reserved.
The study of the spatial configuration of soil, in its complexity, requires an understanding of the interrelations and interactions between the diverse soil constituents, at various levels of organization. Investigations of the spatial arrangement of the mineral and organic components of soil have benefited from the development of techniques for structural analysis. X-ray computed tomography (CT) is a non-destructive and non-invasive technique that has been successfully used for three-dimensional (3D) examination of soil. Valuable information has been obtained by the application of CT for the description and quantitative measurements of soil structure elements, especially of soil pores and pore network features. In many studies, X-ray CT has been used to investigate the hydro-physical characteristics of the soil, in a functional and temporal manner. A dynamic approach has also been utilized in the evaluation of the biotic factor influence on soil. The analysis of soil solid phases, by X-ray CT, has been challenging due to the similar X-ray attenuation of different solid constituents. However, the use of multiple X-ray energy levels has facilitated the discrimination of minerals in soil. The aim of this review and synthesis is to offer a perspective on the major issues related to application of the technique, general attempted solutions and possible directions in the utilization of X-ray CT in soil research. Relevant scanning parameters, procedures for CT image reconstruction, algorithms for the quantification of soil characteristics and results are presented for each type of application.
Methods are described to study the mechanism and rate of sulphide oxidation in water using 35S. These methods were used in the Black Sea together with measurements of O2, H2S, Eh, bacterial biomass and production. Sulphide oxidation proceeds via two main steps: chemical oxidation of sulphide to sulphate and thiosulphate, and the biological oxidation of thiosulphate to sulphate. The rate of sulphide oxidation varied from 0·4–0·9 g H2S/m2 day. The maximum rate of oxidation took place at depths of 150–170 m at Eh –10 to –20 mV, and at a maximum rate of chemosynthesis of 8 mg C/m3/day. Data are given for the biomass of the microflora and its production in the water column. The maximum efficiency of chemosynthesis was 15%. The results are discussed in relation to the budget of the organic matter.
Minimal information has been garnered regarding the spatial distribution of soil water in relation to pores and the soil matrix. Destructive layer-by-layer reconstructions derived from polished section methodology exclude any data of water in the soil. In contrast, microfocus X-ray tube computerized tomography generates images of the internal structure of the soil with a resolution down to 1μm, at the same time creating a visual image of the spatial distribution of water in undisturbed soils.As X-rays pass through the soil, some radiation is absorbed, some is scattered, and some is transmitted. Using advanced microfocus computerized tomography (μCT) which ensures controlled and stable output intensity for X-ray emissions and thus a constant focal spot size and spatial resolution, the resulting pattern of radiation detects to a 0.5% contrast difference. While 2D X-ray imaging is sufficient in many cases, 3D images derived from X-ray irradiation of a soil sample can reveal complex inner structures in more comprehensive format, providing information on the causal connection of water and soil structure.Using the X-ray Feinfocus Y.FOX System and related programmes, two- and three-dimensional images of two different soils (Haplic Luvisol and Stagnic Anthrosol) at field capacity (pF 1.8) have been produced which show films of water which are associated with the pore surfaces. The mean thickness of the water films was 10.6μm in the Stagnic Anthrosol and 3.0μm in the Haplic Luvisol. These results were unexpected in pores >50μm since at field capacity only the adhesive water should be present which would create water films in the range of nanometres. Myriads of colloidal dispersed nanoparticles, detected with dark field microscopy and SEM, seem to be the source of the adhesion and cohesion, causing micro-rheological effects which lead to water films of up to 30μm in pores. Additionally, nanoparticles correlated to the clay content (fine clay) appear to conglomerate in the water films, presumably forming surface protuberances on the films of varying extent.