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The Secret Life of Microbes

CHARLES DARWIN UNIVERSITY

Professorial Lecture Series 5Lecture 26 July 2016

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 GibbSchool of EnvironmentCharles Darwin University

Charles Darwin University

Professorial Lecture Series


Professor Karen GibbSchool of Environment

Tuesday 26 July 2016

Charles Darwin University

Darwin, Northern Territory 0909 Australia

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Published July 2016

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_______________________________________________________ The Secret Life of Microbes 1


Professor Karen GibbPeople 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 birthRise 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


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


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


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.


New HorizonsIn 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’


(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 AskAs 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


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).


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.


In terms of scale, what helps is being able to extract DNA and RNA directly from water, sediment and biota. I mentioned the next-generation 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:

• Microbes and metals• Biota-metal-microbe interactions• Microbes in marine harvest• Extremophiles• Microbial source tracking• Microbes and nutrients.

Microbes and Metals – Coastal MonitoringIn 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).


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,


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.

!


Microbes and Metals – Acid Mine DrainageOne 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 – PolychaetesIn 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.


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.


Biota-Metal-Microbe Interactions – Marine SpongeBucella 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 HarvestPadovan 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,


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 TrackingMicrobial 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


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.


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 NutrientsBacterial 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


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 CommentsEnvironmental 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.


ReferencesBuccella, 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).

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CHARLES DARWIN UNIVERSITY

Professorial Lecture Series 5Lecture 26 July 2016

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 GibbSchool of EnvironmentCharles Darwin University

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