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CHARLES DARWIN UNIVERSITY
The Secret Life of
Microbes
Professor Karen Gibb is an environmental microbiologist with 28 years’ research
experience. She leads the Environmental Chemistry and Microbiology Unit (ECMU),
which is a research and commercial unit at Charles Darwin University. Professor Gibb
and her team investigate the source of contaminants and interpret changes in marine,
estuarine and aquatic environments. ECMU’s research has supported important
improvements in the methodologies and policies that underpin the sustainable
management of marine, estuarine and aquatic systems across Northern Australia.
Government now mandates some of the methodologies developed by ECMU for
environmental monitoring. Professor Gibb has published 120 journal articles, and in 2006
she was awarded, with two colleagues, the Northern Territory Research and Innovation
Tropical Knowledge Award for Research.
Professor Karen Gibb
School of Environment
Charles Darwin University
Professorial Lecture Series 5
Lecture 26 July 2016
Charles Darwin University
Professorial Lecture Series
The Secret Life of Microbes
Professor Karen Gibb
School of Environment
Tuesday 26 July 2016
Charles Darwin University
Darwin, Northern Territory 0909 Australia
T. +61 8 8946 6666
E. [email protected]
W. cdu.edu.au
CRICOS Provider No. 00300K
RTO Provider No. 0373
The Professorial Lecture Series is produced by the Office of Media, Advancement
and Community Engagement (MACE), Charles Darwin University.
Opinions and views expressed in this edition do not necessarily reflect those of
Charles Darwin University.
© Karen Gibb, 2016
Published July 2016
This publication is also available at: www.cdu.edu.au/mace/publications
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The Secret Life of Microbes
Professor Karen Gibb
People generally associate bacteria with food poisoning, unsanitary
conditions and disease, or those unpleasant pink dots on forgotten
food at the back of the fridge. We also tend to think a disease is caused
by a single species that invades its host and overcomes its defences.
There is a long history of misunderstanding and lack of appreciation
about the incredible role of microbes in every aspect of our lives.
Bacteria can be found in water, soil, animals, plants and air. They
are ‘prokaryotes’ and are typically 0.2 to 1 μm long. They can live
in high temperature conditions, frozen ground, acid volcanoes, and
at the bottom of the ocean. They can reproduce by doubling with a
generation time of 20 minutes, or survive for centuries in a resting
stage. One factor in their impact on our lives is their ability to move.
Most bacteria are able to move efficiently to find food or a host, and
they use a few different strategies to achieve movement. The most
common is with the aid of flagella, thin, whip-like structures that
extend from the cell walls of many kinds of bacteria. Some bacteria
have a single, tail-like flagellum or a small cluster of flagella that
rotate in a coordinated fashion, much like the propeller on a boat
engine, to push the organism forward. Bacteria can propel themselves
at a rate of 10 times their body length each second. Many bacteria
also use appendages called pilli (similar to hairs) to move along a
surface. These pilli bind receptors and pull a bacterium forward when
retracted. Bacteria use chemical cues from the environment, that is
chemotaxis, to move to useful places. A bacterium tracking down a
chemical stimulant (such as a nutrient) moves in a way known as
‘random walking’.
The discipline of ‘microbial ecology’ is relatively new and is revealing
some incredible knowledge about microbes and the environment. To
appreciate how far we have come in this new discipline, including
new knowledge close to home, I would like to first highlight some of
the major paradigm shifts and enabling technologies that have paved
the way.
The English physician and grandfather of Charles Darwin, Erasmus
Darwin (1731–1802), a natural philosopher, inventor and poet, wrote
about microbes. As an aside, Darwin was also a founding member of
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the Lunar Society of Birmingham, a discussion group of pioneering
industrialists and natural philosophers including Josiah Wedgwood,
Matthew Boulton, James Watt (Boulton and Watt steam engines),
Joseph Priestley (clergyman, chemist and credited with the discovery
of oxygen) to name a few. The name arose because the society
would meet during the full moon, the extra light making the journey
home easier and safer in the absence of street lighting. They called
themselves ‘lunarticks’. Darwin’s most important scientific work,
Zoonomia (1794–1796), contains a chapter that foreshadowed the
modern theory of evolution. Erasmus Darwin's works were read and
commented on by his grandson. Erasmus Darwin's final long poem,
The Temple of Nature, was published posthumously in 1803 and is
considered his best poetic work. The poem was originally titled The
Origin of Society. It centres on his conception of evolution and traces
the progression of life from microorganisms to civilised society.
‘Hence without parent by spontaneous birth
Rise the first specks of animated earth.’
(An excerpt from Canto I. l. 227, The Temple of Nature by
Erasmus Darwin http://knarf.english.upenn.edu/Darwin/
templetp.html)
Darwin wrote a number of ‘additional notes’ to the poem including
one on the Spontaneous Vitality of Microscopic Animals:
‘Experimental facts.
III. By the experiments of Buffon, Reaumur, Ellis, Ingenhouz,
and others, microscopic animals are produced in three or
four days, according to the warmth of the season, in the
infusions of all vegetable or animal matter. One or more of
these gentlemen put some boiling veal broth into a phial
previously heated in the fire, and sealing it up hermetically or
with melted wax, observed it to be replete with animalcules in
three or four days.
And …
Besides this green vegetable matter of Dr. Priestley, there is
another vegetable, the minute beginnings of the growth of
which Mr. Ellis observed by his microscope near the surface
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of all putrefying vegetable or animal matter, which is the
mucor or mouldiness; the vegetation of which was amazingly
quick so as to be almost seen, and soon became so large
as to be visible to the naked eye. It is difficult to conceive
how the seeds of this mucor can float so universally in the
atmosphere as to fix itself on all putrid matter in all places.’
(http://knarf.english.upenn.edu/Darwin/templetp.html.)
I don’t see that Darwin is referring to spontaneous generation, but
some commentators suggest that he is: I am not convinced. I think, in
fact, he may be describing biofilms.
Well into the 1800s scientists believed that microbes originated
from nothing in a process called ‘spontaneous generation’. In 1861,
however, Pasteur disproved this notion by showing that nutrient
broth would only become cloudy with the growth of bacteria if it were
exposed to dust in the air carrying living bacteria (Figure 1). In other
words, the bacteria did not come into existence from nothing.
Figure 1: Pasteur’s swan neck flasks that helped disprove the notion of
spontaneous generation. (http://www.eeescience.utoledo.edu)
In 1857, Pasteur proposed the Germ Theory of Disease, which proposed
that bacteria or other microbes caused several human diseases. Until
then, people thought spirits, bad air or the faulty character of the
individual, caused diseases. In 1876, Robert Koch provided evidence
in support of the Germ Theory by showing that anthrax is caused
by Bacillus anthracis. Koch also developed the first methods to grow
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bacteria in pure cultures as colonies on potato slices. In 1890, Koch
postulated four criteria designed to establish a causative relationship
between a microbe and a disease. Koch’s postulates have played an
important role in microbiology, yet they have major limitations; for
example, Koch was well aware that in the case of cholera, the causal
agent – Vibrio cholerae – could be found in both sick and healthy
people, invalidating his first postulate.
More recently, modern nucleic acid-based microbial detection methods
have made Koch’s original postulates even less relevant. These
nucleic acid-based methods make it possible to identify microbes
that are associated with a disease, but in many cases the microbes
are uncultivable. Also, nucleic acid-based detection methods are very
sensitive, and they can often detect the very low levels of viruses that
are found in healthy people without disease.
There is a prevailing but mistaken view that microorganisms isolated
in pure culture from an environment represent the numerically
dominant and/or functionally significant species in that environment.
In fact, microorganisms isolated using standard cultivation methods
are rarely numerically dominant in the communities from which they
are obtained, instead they are isolated by virtue of their ability to
grow rapidly into colonies on high-nutrient artificial growth media,
typically under aerobic conditions, at moderate temperatures. These
easily isolated organisms are estimated to constitute less than one
per cent of all microbial species. This is known as the great plate
count anomaly. It was so named because counts of cells obtained via
cultivation are orders of magnitude lower than those directly observed
under the microscope (Hugenholtz, 2002).
The significance of this is that most of what we know about microbiology
comes from the less than one per cent of microbes that are relatively
simple to grow on agar plates. It seems unlikely that this handful of
organisms can be representative of the approximately 5000 validly
described prokaryotic species.
Having said that, there is still a place for culturing organisms as a way
to study their morphology, biochemistry and interactions with other
compounds. Using this more conventional approach, we showed
fungi outcompete bacteria under increased uranium concentration
in culture media (Mumtaz et al., 2013). Soil samples collected
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from the Ranger Uranium Mine in the Northern Territory and with
different concentrations of uranium were plated on to nutrient media
containing no (0 ppm), low (3 ppm), medium (250 ppm), high (600
ppm) and very high (1500 ppm) uranium concentrations. Bacteria
grew on all plates except for the very high uranium concentrations,
where only fungi were recovered. The dominant cultivable bacteria
belonged to the genus Bacillus. Fungi were dominant on plates
with very high uranium concentrations and included Aspergillus,
Cryptococcus and Penicillium. These findings indicate that fungi can
tolerate very high concentrations of uranium and are more resistant
than bacteria. Bacteria and fungi isolated from Ranger soils with high
concentrations of uranium may have uranium-binding capability
and hence the potential for uranium bioremediation. Some of the
wonderfully coloured bacteria isolated from the Ranger uranium soil
sites are shown in Figure 2.
Figure 2: Some of the bacteria isolated from Ranger Uranium Mine soils.
The reality, however, is that if more than 99 per cent of microorganisms
in the environment are uncultivable, how representative are cultivated
microorganisms of prokaryotic diversity as a whole? To answer these
questions, we need a framework for placing prokaryotic species
and genera in a broader evolutionary context. The problem is that
microbiologists have not, with some exceptions, successfully embraced
evolutionary biology principles in their thinking, experimentation or
writing (Woese, 1987).
Moreover, the failure to determine evolutionary relationships seemed
to generate the feeling that it was not important to do so. Bacterial
evolution was all but forgotten and when a more powerful approach
for measuring evolutionary relationships, that is DNA sequencing,
became available in the mid 1950s, it was largely ignored by
microbiologists for more than a decade.
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New Horizons
In 1987, Carl Woese showed that a subunit of the small subunit
ribosomal RNA gene, also known as 16S rRNA, in bacteria was an
excellent molecular clock and useful for measuring relationships
between bacteria. The 16S rRNA genes are present in all bacteria (a
subunit of bacterial ribosomes), they are functionally constant, and
different positions in the sequence change at different rates allowing
most phylogenetic relationships to be measured. Although there are
now countless papers that use the 16S rRNA gene to measure genetic
relationships, it would be fair to say very few even now are tackling
significant evolutionary questions.
When high throughput massive parallel sequencing technologies
emerged using the 16S rRNA ‘tag’ – techniques such as GS FLX, the
latest pyrosequencing platform by 454 by Roche Diagnostics – it could
generate 400 Megabases of DNA (400 million nucleotides) in a 10-hour
run with a single machine. A nucleotide is a unit of DNA (Figure 3).
This chemistry was discontinued in 2013 and many people now use
MiSeq Technology by Illumina, which can generate up to 4 Gigabases
of DNA (4 billion nucleotides) in less than a day.
Thanks to these DNA technologies, microbiologists have emerged from
the world of culture plates and microscopes. What has resulted is the
relatively new discipline of microbial ecology. Researchers in this field
explore an incredibly diverse range of microbial landscapes. Much of
the technological drive has come from human research and the most
famous example is the ‘Human Microbiome Project’ (HMP) (http://
commonfund.nih.gov/hmp/index). Associations are being measured
between human health and our microbiome, and researchers suggest
our microbiome is linked to gastrointestinal disorders, acne, obesity,
moods, behavior and even our thoughts (Bakhtiar et al., 2013),
although I’m not sure what Robert Koch (Koch’s postulates on cause
and effect) would think about some of those claims. Statements such
as ‘Your body has 10 times as many microbe cells as human cells’
could be dismissed as trite, but at one level they are powerful statistics
that can be used to reinforce the understanding that microbes are
more than just harbingers of disease. For about US$97 Americans
can have their gut microbiota measured and for that you get the
taxonomy of your gut microbes and an assessment of how your gut
microbes compare to others. This program is called ‘American Gut’
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(http://humanfoodproject.com) and it is generating a huge dataset
for researchers because the subscribers also provide important
supporting data such as gender, age, weight, smoking status, alcohol
consumption and other metrics.
Figure 3: Structure of a nucleotide – the units of DNA.
Knowing the Questions to Ask
As powerful as our new tools are, microbiologists can be limited in
their understanding of ecosystem complexities, and this can mean
the tools are not deployed to their best effect. We’re generally not
good at asking the important evolutionary questions, and it is only
now we are starting to ask. When the DNA sequencing tools first
came out, researchers sampled every interesting environment from
deserts to hydrothermal vents to saline lakes to peat bogs to soils
permanently buried beneath snow. This resulted in an avalanche of
_______________________________________________________ The Secret Life of Microbes 7
descriptive microbial community papers with little appreciation for,
or accommodation of, ecological questions or experimental design. I
recall an international microbial ecology conference in the early 2000s
where someone reported DNA and microbial community analysis of
the English Channel – sampled with no particular question in mind.
The room was packed and it was all people could talk about. Next
session in a small side room was a lovely talk on the ecology of a lake
where more conventional bacterial analysis techniques were used
– but asking really interesting ecological questions about the lake’s
hydrology and nutrient cycling. It was so interesting and was the only
ecological talk there – and the woman apologised for the absence of
DNA analysis! I realised then and there that I didn’t want to be in that
first group.
Environmental microbiology is the ecology of microorganisms
and how they relate and function with one another and with their
environment. Microorganisms control global biogeochemical cycling
by virtue of their role in the cycling of nitrogen, carbon and sulfur.
The immensity of microorganisms’ production is such that, even
in the total absence of eukaryotic life, these processes would likely
continue unchanged (Yarza et al., 2014). This leads me to a branch of
environmental microbiology known as geomicrobiology and microbial
geochemistry (GMG). GMG did not exist 40 years ago, but it is an
incredibly important interdisciplinary discipline because it has shown
that Earth’s history is completely interwoven with the evolution of
microbes (Druschel and Kappler, 2015). Some examples of GMG
research include studies on iron and sulfur chemistry in ancient
seas, cycling of carbon and nitrogen, and how microbes mobilise
metal, radionuclides and organic pollutants, thereby making them
more potentially damaging. Microbes play critical roles in processes
affecting water quality and agriculture. Their role in nitrogen cycling
is key to understanding greenhouse gas levels, nutrients for crops, and
water quality issues including harmful algal blooms. Bacteria have an
uncanny capacity to exploit an opportunity, for example the bacteria
genus Vibrio, which includes the agent of cholera, can exploit poor
quality water by adhering to the particles and using the nutrients on
those particles. What is more, they themselves bloom in the presence
of chitin, a compound found in diatoms and the tiny zooplankton often
present in poor quality water (Watkins and Cabelli, 1985).
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But I should step back for a moment to look at how new fields such as
GMG arise. Kenneth Nealson, a scientist in this field, reflected on this
(Nealson, 2015) using his own experience as the guide. He suggested
that new fields arose when people from these various disciplines had
an opportunity to be in contact with each other. This might seem
obvious but, for example, during my postdoctoral fellowship at the
University of Kentucky, the department was so big I pretty much just
saw and spoke to people in my own discipline. This is very common.
In Nealson’s view and in relation to GMG, the geochemists identified
the problems but lacked the methods to address them. In contrast,
the microbiologists did not have the questions, but had the techniques
(methods). This may seem harsh with respect to the microbiologists
but actually mirrored my own thoughts at the conference mentioned
above. Nealson offered a third key ingredient to the launching of
GMG based on his own experience. In his case a marine geochemist
reflected after a seminar that iron and manganese geochemistry in the
ocean was not well understood, and he suspected bacteria drove the
processes. This chance comment drove his future research program.
These days scientists are at an advantage if they do interdisciplinary
research. This bodes well for our own university where most schools
and research institutes comprise people from different disciplines,
fostering conversations and new ideas.
As I have mentioned, the new DNA-based techniques allow us to study
microbial ‘landscapes’. As a result, we have discovered the incredible
biodiversity and function of bacteria in even the most inhospitable
environments. This relatively new field of research is providing
exciting insights into how bacteria form ‘cities’ that communicate and
respond to changes in the environment, and their role as early warning
sentinels of change. I will also draw on our own research to reveal
how bacteria are helping us to understand our local environment,
with a particular focus on Darwin Harbour.
Our research at Charles Darwin University focuses on microbiology
in a stressful environment. This requires us to understand other
disciplines well enough to know what questions to ask. We focus
on what is natural, that is: what is there normally; and what is not
natural and why? Challenges include scaling up to ecological relevant
scale and being able to contribute at the landscape scale. Another
stressor is the complexities associated with pollutant interactions in
the environment.
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In terms of scale, what helps is being able to extract DNA and RNA
directly from water, sediment and biota. I mentioned the nextgeneration technologies previously; we were among the first to use
these in Australia and were one of the Australian Genome Research
Facility’s first 454 next-generation sequencing client.
I will now talk about our research in the natural environment and
some of the challenges this brings. Our research falls into a range of
categories:
•
•
•
•
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Microbes and metals
Biota-metal-microbe interactions
Microbes in marine harvest
Extremophiles
Microbial source tracking
Microbes and nutrients.
Microbes and Metals – Coastal Monitoring
In 2007, when we started our ARC L-P funded work on Coastal
monitoring using metal resistant microbes, we had been influenced by
reports from the literature where microbiologists dissolved metals in
nutrient agar media and ‘fed’ marine microbes. They found changes
in the microbiology community associate with metals, and the higher
the metal concentration, the less diverse the community. This was
taken to show that pollution from metals reduced biodiversity;
it was a common theme and one we planned to follow. Our team,
however, included environmental chemists and they raised the
issue of bioavailability. Researchers have struggled for decades with
concepts and definitions of bioavailability (Semple et al., 2004). Thus,
just the presence of substances of concern is not sufficient; harmful
interaction with a receptor must be possible as well. Because toxic
effects require that an organism takes up the contaminant, the extent
to which substances are bound to soil particles or are available to cause
harm needs to be considered. This was relatively new for biologists
– certainly for microbiologists, but chemists had known about it for a
long time. It also borrowed heavily from toxicology. Figure 4 captures
the concepts of bioavailable and bio-accessible, but also shows that
microbes are assumed to access pollutants dissolved in pore water
(the water between soil and sediment particles).
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Figure 4: Conceptual diagram of bioavailability of pollutants in sediment
(Semple et al., 2004). Used with permission.
We had planned to expose microbes to media plates infused with
different concentrations of dissolved metals. The levels of metals we
planned to use would reflect total metal levels in the environment – in
this case Melville Bay near Gove associated with Rio Tinto’s alumina
facility.
This diagram suggested, however, that microbes would only be
responsive to pore water metal levels. This was a concern from the
point of view of the original premise of the work. Pore water metal
levels in Melville Bay were actually very low and we thought unlikely
to affect microbes at all.
In fact, what we found was that microbes did not change in response
to pore water metal levels or easily extracted levels; they changed
in response to bioavailable and total metal levels (Figure 5). Their
responsiveness to HCl-extractable metals told us a few things: first,
they were responsive to metal levels that were potentially the same
as those experienced by mammals living in this environment; second,
they were a valid surrogate for higher organisms in this instance –
and therefore potential early warning sentinels of change; and third,
_______________________________________________________ The Secret Life of Microbes 11
these communities were a potential source of bio-assessment tools for
routine monitoring (Cornall et al., 2013).
The metal profiles of samples and the bacteria community composition
gave similar patterns, that is, the high-impact sites near the discharge
clustered together whether or not metals or bacteria were measured,
so too did other measures such as sulphur levels. Metals and sulphides
can occur together in low oxygen environments, so the problem is:
what are the bacteria responding to? We addressed this by identifying
the bacteria associated with total metals, those associated with
bioavailable metals and those associated with physicochemistry data
such as sulphur levels. Then we looked at the intersection sets of these
three and only took bacteria that were in the intersection between
the bioavailable and total metals, excluding those associated with
physicochemistry. So this subset gave us potential targets for routine
monitoring, with a particular focus on metals.
Figure 5: PCO plots of OTU bacteria resemblance matrix data overlaid with
vectors corresponding to normalised data for (a) porewater metals, (b) easily
extractable metals, (c) HCl-extractable metals, and (d) total extractable metals.
Each metal included in the analysis is represented by a vector that extends
towards the direction in which the metal has the greatest correlation with
community structure. Longer vectors indicate stronger correlations.
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!
Microbes and Metals – Acid Mine Drainage
One of the most difficult environmental issues for the mining industry
is acid mine drainage (AMD), which occurs when metal sulphides,
such as pyrite, are solubilised by oxidative dissolution leading to the
production of sulfuric acid. This sulfuric acid solubilises other metals
present in the ore body, which are added to the drainage product. The
acidic, metal-rich discharge into surface and groundwater can lead to
significant environmental damage. We studied AMD through an ARC
L-P funded project Managing acid mine drainage in northern Australia
using microbial mats. We identified bacteria and fungi associated
with AMD at Rum Jungle. Rum Jungle mine is located about 105 km
by road south of Darwin, and uranium was mined from open pits
from 1954 to 1958, while copper was extracted until 1964. The soil
contains sulphide minerals, pyrite, chalcopyrite, chalcocite, covellite
and galena. Waste rock dumps at Rum Jungle were covered with clay
and gravel during 1983–1986. The clay caps have cracked and water
can now enter during the wet season, which has led to the generation
of AMD (Streten-Joyce et al., 2013). We measured the chemical
composition and bacteria communities in acid and metalliferous
drainage from this area and showed they were dependent on season.
Mats prepared in the laboratory using cultured components that we
had identified from the field successfully removed some metals such
as arsenic and cadmium.
Biota-Metal-Microbe Interactions – Polychaetes
In a study of the bacterial community associated with the marine
polychaete Ophelina sp. 1, Neave et al. (2012) showed that the
bacteria community was altered by copper and zinc contamination in
sediments. Tolerant species of polychaete worms can survive in polluted
environments using various resistance mechanisms. One aspect of
resistance not often studied in polychaetes is their association with
symbiotic bacteria, some of which have resistance to metals and may
help the organism to survive. We used ‘next generation‘ 454 sequencing
of bacterial 16S rRNA sequences associated with polychaetes from
a copper and zinc-polluted Cullen Bay marina and from the control
Dinah Beach site to determine bacterial community structure. We
found changes in the bacteria at the polluted site, including increases
in the abundance of bacteria from the order Alteromonadales. These
changes in the bacteria associated with polychaetes may be relatively
easy to detect and could be a useful indicator of metal pollution.
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Three treatment types were analysed in this study: bacteria associated
with washed polychaetes, unwashed polychaetes and in the sediment.
Results for the unwashed polychaete treatment were expected
to ascertain which bacteria were associated with the polychaete
epidermis or mucus layer, and this is where the biggest difference
between Cullen Bay and Dinah Beach was observed (Figure 6). The
bacteria that were detected in the washed polychaete treatments were
relatively similar at the two sites; this was surprising given that tightly
associated bacteria were expected to be the niche involved in metal
detoxification processes (Figure 6). Others have reported, however,
that mucus secretion and bacteria within the mucus appear to have
detoxifying properties. It is likely that our washing treatment removed
most of the mucus from the worms, therefore the large bacterial
changes may have been related to bacteria within the mucus layer,
and these bacteria may be involved in metal detoxification. The
sediment bacterial community was relatively similar at both Cullen
Bay and Dinah Beach, despite the large differences in copper and zinc
concentrations in the sediments. The copper and zinc concentrations
were much more influential on the bacterial community associated
with Ophelina sp.1, suggesting that the associated bacteria are a more
sensitive indicator of pollution.
Figure 6: Multi-dimensional scaling (MDS) plot showing similarities among
the bacterial communities associated with polychaetes and sediments. The
metals that were correlated with the bacterial community patterns (Spearman’s
correlation > 0.6) were overlaid on to the plot.
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Biota-Metal-Microbe Interactions – Marine Sponge
Bucella et al. (2014) showed that the tropical marine sponge,
Halichondria phakellioides, from Darwin Harbour contained high
concentrations of molybdenum. A rod-like bacterium extracellular in
sponge tissue was observed using transmission electron microscopy.
Molybdenum was located within these bacteria, but not in sponge cells.
This is the first report of the trace element molybdenum localised in a
sponge bacterial symbiont. Many different bacterial symbionts were
identified in the sponge by sequence analysis, so the identity of the
molybdenum-accumulating bacterium could only be inferred.
Microbes in Marine Harvest
Padovan et al. (in review) took multiple approaches to measuring
seafood quality in Darwin Harbour in collaboration with Larrakia
elders. In this study, metal and metalloid concentrations and pathogens
were measured in shellfish at various locations in a tropical estuary,
including sites impacted by sewage and industry. Oyster, mangrove
snails and mud snails did not exceed Australian and New Zealand
Food Standards maximum levels for Cd, Pb, or estimated inorganic
as at any site, although Cu concentrations in oysters and mud snails
exceeded generally expected levels at some locations. Bacterial
community composition in shellfish was species-specific regardless
of location, and different to the surrounding water and sediment.
In the snails Telescopium telescopium, Terebralia palustris and
Nerita balteata, some bacterial taxa differed between sites, but not
in Saccostrea cucullata oysters. The abundance of potential human
pathogens was very low and pathogen abundance or diversity was
not associated with site classification, that is sewage impact, industry
impact and control.
Extremophiles – You Call This a Niche?
In a study by Tracy et al. (2010) we measured the microclimate
and limits to photosynthesis in a diverse community of hypolithic
cyanobacteria in Northern Australia. Hypolithic microbes, primarily
cyanobacteria, inhabit the highly specialised microhabitats under
translucent rocks in extreme environments. We analysed hypolithic
cyanobacteria found under three types of translucent rocks (quartz,
_______________________________________________________ The Secret Life of Microbes 15
prehnite and agate) in a semiarid region of tropical Australia. We
investigated the photosynthetic responses of the cyanobacterial
communities to light, temperature and moisture in the laboratory,
and we measured the microclimatic variables of temperature and
soil moisture under rocks in the field over an annual cycle. We also
used molecular techniques to explore the diversity of hypolithic
cyanobacteria in this community and their phylogenetic relationships
within the context of hypolithic cyanobacteria from other continents.
Based on the laboratory experiments, photosynthetic activity required
a minimum soil moisture of 15 per cent (by mass). Peak photosynthetic
activity occurred between approximately 8°C and 42°C, though
some photosynthesis occurred between −1°C and 51°C. Maximum
photosynthesis rates also occurred at light levels of approximately
150–550 μmol m−2 s−1. We used the field microclimatic data in
conjunction with these measurements of photosynthetic efficiency
to estimate the amount of time the hypolithic cyanobacteria could
be photosynthetically active in the field. Based on these data, we
estimated that conditions were appropriate for photosynthetic activity
for approximately 942 h (∼75 days) during the year (Figure 7). The
hypolithic cyanobacteria community under quartz, prehnite and
agate rocks was quite diverse both within and between rock types.
We identified 115 operational taxonomic units (OTUs), with each rock
hosting 8–24 OTUs. A third of the cyanobacteria OTUs from Northern
Australia grouped with Chroococcidiopsis, a genus that has been
identified from hypolithic and endolithic communities found in the
Gobi, Mojave, Atacama and Antarctic deserts. Several OTUs identified
from northern Australia have not been reported to be associated with
hypolithic communities previously.
Microbial Source Tracking
Microbial source tracking is an area of research in which multiple
approaches are used to identify the sources of elevated bacterial
concentrations in recreational lakes and beaches (Neave et al., 2014).
At our study location, Darwin Harbour, water quality in the harbor
is generally good, but dry season beach closures due to elevated
Escherichia coli and enterococci counts are a cause for concern.
The sources of these high bacteria counts are currently unknown. To
address this, we sampled sewage outfalls, other potential inputs, such
as urban rivers and drains, and surrounding beaches (Figure 8), and
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used genetic fingerprints from E. coli and enterococci communities,
fecal markers and 454 pyrosequencing to track contamination
sources. A sewage effluent outfall (Larrakia discharge) was a source
of bacteria, including fecal bacteria that impacted nearby beaches.
Two other treated effluent discharges did not appear to influence sites
other than those directly adjacent. Several beaches contained fecal
indicator bacteria that likely originated from urban rivers and creeks
within the catchment. Generally, connectivity between the sites was
observed within distinct geographical locations and it appeared that
most of the bacterial contamination on Darwin beaches was confined
to local sources.
Figure 7: Microclimate temperature under translucent rocks that supported
hypolithic cyanobacteria. Data are means of hourly temperatures recorded
immediately under several rocks between 3 June 2006 and 10 June 2007.
The broken line represents the maximum temperature at which these
cyanobacterial communities can photosynthesise. Hypolithic temperatures
exceeded this maximum for 600 h on 131 days. Temperatures never dropped
below the minimum for photosynthesis. Black bars at the bottom of the graph
indicate days when soil water content was above 0.15 m3 m-3, the threshold
for photosynthetic activity of hypolithic communities. Water content exceeded
this threshold on 75 days.
_______________________________________________________ The Secret Life of Microbes 17
Figure 8: Sites overlaid with an indication of fecal indicator bacteria (FIB) counts
per 100 mL of water. Diamonds are the sewage discharge sites, squares are other
inputs, triangles are the beaches, circles are Rapid Creek and inverted triangles
are Lake Alexander. The control site 30 “Wagait Beach” west of Darwin Harbour
is not shown and had a FIB count of <100.
Microbes and Nutrients
Bacterial community composition can change as a result of increased
nutrient loads and may be useful for assessing ecosystem health in
estuaries. But the ability to understand how bacterial communities
respond to increased nutrient concentrations is limited by the paucity of
community level bacterial base data, in particular for tropical estuaries.
Our aim was to describe and compare the bacterial community in the
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water column and sediments across tropical tidal creeks in Darwin
Harbour. We assessed the relationship between communities and
increased nutrient loads, comparing sites with sewage effluent inputs
to control sites. In this tropical estuary, bacterial species richness
and diversity in water increased with increased nutrient load. This
result suggests that there is an untapped resource of bacteria that
should be explored as potential water quality indicators for receiving
environments. We showed that taxa such as Aeromonas, Azomonas
and various cyanobacteria have potential as water quality indicators,
not just for public health but also as measures of ecosystem health.
The close association between bacteria community composition and
elevated nutrients in estuaries could be exploited to select a range of
water quality indicators (Gibb et al., 2016).
Concluding Comments
Environmental microbiology or microbial ecology is a rich new
discipline that embraces other disciplines to provide depth and
diversity. We have shown just a subsample of the interesting projects
we have undertaken, and we have more underway and planned that I
did not have time to cover in this lecture. It is an exceedingly interesting
area of research but, perhaps most importantly, our emergence from
the laboratory has made microbiology more accessible and visible to
environmental scientists who often overlook the smallest creatures. It
bodes well for future collaborative research to address environmental
pressures and climate change. Already we see water quality lessons in
our warm marine waters that serve as a warning to other places that
might eventually see similar water temperatures.
I hope I have shown that microbes are not only ubiquitous but that
they also drive the global biosphere and should not be forgotten in
environmental science.
_______________________________________________________ The Secret Life of Microbes 19
References
Buccella, C., B. Alvarez, K. Gibb and A. Padovan (2014). A rod-like bacterium
is responsible for high molybdenum concentrations in the tropical sponge
Halichondria phakellioides. Marine and Freshwater Research, 65(9): 838–48.
Cornall, A. M., S. Beyer, A. Rose, C. Streten-Joyce, K. McGuinness, D. Parry and K.
Gibb (2013). HCL-extractable metal profiles correlate with bacterial population
shifts in metal-impacted anoxic coastal sediment from the wet/dry tropics.
Geomicrobiology Journal, 30(1): 48–60.
Donlan, R. M. (2002). Biofilms: Microbial Life on Surfaces. Emerging Infectious
Diseases, 8(9): 881–90. http://doi.org/10.3201/eid0809.020063
Druschel, G. and A. Kappler (2015). Geomicrobiology and Microbial Geochemistry.
Elements, 11: 389–94
Gibb, K., M. Kaestli, J. Smith and K. McGuinness (2016). Broadening the Targets
for Microbial Water Quality Water e-Journal Online journal of the Australian
Water
Association
http://www.awa.asn.au/AWA_MBRR/Publications/
Water_e-Journal/02_PDF_Water_Quality_Gibb.aspx.
Hugenholtz, P. (2002). Exploring prokaryotic diversity in the genomic era. Genome
Biology, 3(2): 1–8
Lupp, Claudia (2009). Microbial oceanography. Nature, 459(7244): 179.
Mumtaz, Saqib, C. Streten-Joyce, D. Parry, K. McGuinness, P. Lu and K. Gibb (2013).
Fungi outcompete bacteria under increased uranium concentration in culture
media. Journal of Environmental Radioactivity, 120: 39–44.
Nealson KH (2015) Geomicrobiology and Microbial Geochemistry: A View from the
Past. Elements, 11: 384–5
Neave, M. J., C. Streten-Joyce, C. J. Glasby, K. A. McGuinness, D. L. Parry and
K. S. Gibb (2012). The bacterial community associated with the marine
polychaete Ophelina sp. 1 (Annelida: Opheliidae) is altered by copper and zinc
contamination in sediments. Microbial ecology, 63(3): 639–50.
Neave, M., H. Luter, A. Padovan, S. Townsend, X. Schobben and K. Gibb (2014).
Multiple approaches to microbial source tracking in tropical northern
Australia. MicrobiologyOpen, 3(6): 860–74.
Semple, K. T., K. J. Doick, K. C. Jones, P. Burauel, A. Craven and H. Harms (2004).
Peer reviewed: defining bioavailability and bioaccessibility of contaminated
soil and sediment is complicated. Environmental Science & Technology,
38(12): 228A–231A.
Streten-Joyce, C., J. Manning, K. S. Gibb, B. A. Neilan and D. L. Parry (2013).
The chemical composition and bacteria communities in acid and metalliferous
drainage from the wet–dry tropics are dependent on season. Science of the
Total Environment, 443: 65–79.
Syeda M. Bakhtiar1, Jean Guy LeBlanc, Emiliano Salvucci, Amjad Ali, Rebeca Martin,
Philippe Langella, Jean-Marc Chatel, Anderson Miyoshi, Luis G. BermúdezHumarán and Vasco Azevedo. Perturbation of the Human Microbiome as a
Contributor to Inflammatory Bowel Disease. FEMS Microbiol Lett 342:10–17).
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Tracy, C. R., C. Streten Joyce, R. Dalton, K. E. Nussear, K. S. Gibb and K. A. Christian
(2010). Microclimate and limits to photosynthesis in a diverse community of
hypolithic cyanobacteria in northern Australia. Environmental microbiology,
12(3): 592–607.
W. D. Watkins and V. J. Cabelli (1985). Effect of Fecal Pollution on Vibrio
parahaemolyticus. Densities in an Estuarine Environment. Applied and
Environmental Microbiology, May 1985, Vol. 49, No. 5, pp. 1307–1313,.
Woese, C.R. (1987). Bacterial evolution. Microbiol Rev, 1987, 51:221–71. Yarza, P. et al. (2014). Uniting the classification of cultured and uncultured bacteria
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_______________________________________________________ The Secret Life of Microbes 21
CHARLES DARWIN UNIVERSITY
The Secret Life of
Microbes
Professor Karen Gibb is an environmental microbiologist with 28 years’ research
experience. She leads the Environmental Chemistry and Microbiology Unit (ECMU),
which is a research and commercial unit at Charles Darwin University. Professor Gibb
and her team investigate the source of contaminants and interpret changes in marine,
estuarine and aquatic environments. ECMU’s research has supported important
improvements in the methodologies and policies that underpin the sustainable
management of marine, estuarine and aquatic systems across Northern Australia.
Government now mandates some of the methodologies developed by ECMU for
environmental monitoring. Professor Gibb has published 120 journal articles, and in 2006
she was awarded, with two colleagues, the Northern Territory Research and Innovation
Tropical Knowledge Award for Research.
Professor Karen Gibb
School of Environment
Charles Darwin University
Professorial Lecture Series 5
Lecture 26 July 2016