Soil Bacteria
Mud, glorious mud…
The earth beneath our feet has taken billions of years to evolve, but this most precious resource will have to be managed if we want life on Earth to survive. Scientists today are using new DNA sequencing methods to understand the composition of microbial communities in the soil – not just to save billions of dollars and reduce atmospheric and water pollution, but also to ensure the soil continues to feed us in future...
"Nothing endures but the land," said Chris Guthrie in the well-known Scottish novel Sunset Song, set in the countryside near Aberdeen. William Blake saw "a world in a grain of sand." But even though Chris had a sense of history and Blake was a visionary poet, they could not see that inside every single gram of soil there are billions of bacteria that first colonised the Earth about four billion years before humans appeared. And according to Professor Jim Prosser, Chair in Environmental Microbiology at the School of Biological Sciences in the University of Aberdeen, a gram of soil also contains up to a million different species of bacteria – plus many other microbes which scientists have only discovered in the last two decades.
Prosser's research focuses on nitrification, studying how soil-nitrifying bacteria and archaea break down ammonia to nitrate – a process which results in enormous economic losses of ammonia-based fertilisers and production of greenhouse gases. His aim is to "understand, predict and manipulate the factors that determine the composition of the nitrifier community and its impact on function," or in other words what helps the soil do its business. This involves "looking at the ecology of soil-nitrifying bacteria and archaea" and "designing strategies to reduce their activity and thereby optimise the efficiency of fertiliser use." This requires analysis of samples of soil using the latest genetic sequencing techniques to understand how conditions affect nitrifiers and promote or inhibit crop growth, so farmers can decide what types of ammonia-based fertilisers work best in specific conditions, depending on the types of nitrifiers in the soil.
To paraphrase one of Prosser's colleagues, describing the relative importance of this branch of science: "If the last nitrifier was gasping for air on the beach, it would be catastrophic for the planet and much more significant than the extinction of the panda." And he could have added that the panda and most other species on Earth would be wiped out soon after the last nitrifier, because it is such an integral part of the food chain.
To explain why all this matters, Prosser begins by describing the amazing complexity and diversity of the soil – one gram of which contains as many bacteria as human beings on the planet, distributed in similar patterns, with clusters of bacteria in 'hot spots' like people in cities, and colonising roots that leak nutrients in the same way that villages develop along rivers and railway lines. There are also thousands of structurally complex micro-environments in every gram of soil, with pores and tunnels only 10 microns wide.
The mass of the bacteria is also important, says Prosser. In one acre of typical farmland, the mass of the bacteria is equivalent to roughly 15 sheep. Sheep obviously have a major impact on the environment (e.g., urinating on their favourite patches of land, creating hot spots of ammonia, fuelling nitrifiers and increasing production of the greenhouse gas nitrous oxide), but the bacteria are overlooked because they can't be seen.
Diversity is also a critical factor, while bacteria also evolve very quickly – if they perform different functions, they tend to survive, whereas if they are competing to perform the same function, weaker specimens tend to die off. The distribution of bacteria is also similar to humans on Earth, and the chances of meeting a "competitor" are roughly the same – such as Vikings meeting Samurai. And according to Prosser, bacteria develop their genetic differences faster than humans.
Prosser also explains that using the word "species" for bacteria is controversial, since the traditional definition states that members of a species can reproduce sexually with each other, but not (or only very infrequently) with members of other species. Although bacteria do reproduce sexually – a chromosome divides before cell division, with daughter chromosomes segregating between the two daughter cells – genetic variability arises through mutations and, importantly, through transfer of genes through virus infection or by exchanging plasmids (a common mechanism for the spread of antibiotic resistance). Consequently, there are huge variations within the same species – for example, E. coli comes in a number of flavours, with different members of the species containing different genes performing different functions. The different members of a 'species' have a core of genes common to all, a 'shell' of genes common to some but not all, and a large 'cloud' of genes present in just a few variants. The functions of these 'cloud' genes can be critical for behaviour, pathogenicity and ecology and may give a particular variant or strain unique characteristics, which make a strain useful in the quest to solve functional problems such as boosting production or improving resistance to certain diseases.
"There are also orders of magnitude more diversity in bacteria than any other species," says Prosser. Ten grams of a typical 'garden' soil will contain a million different traditional 'species,' whilst a few metres away the bacterial community may be very different. Communities in soil from a hundred metres further away will be even more different – even though, on the surface, we see nothing different.
So why does diversity matter? The composition of the soil bacterial communities is very complex and varies dramatically in different locations, but the ability of microbes to perform different functions is vital to the health of the planet. A single gram of soil will contain bacterial species capable of breaking down the majority of naturally-occurring compounds and, in total, the soil bacterial community can degrade all but a few man-made compounds. If the bacteria could not degrade them, we would be up to our necks in these compounds, says Prosser, pointing out that increasing awareness of the global accumulation of plastics shows what can happen when non-degradable compounds are mass-produced. Soil is also a vast reservoir of bacteria with potentially useful characteristics, including the production of powerful antibiotics, partly because they need them to defend themselves against other organisms.
One revolution after another
Over the last 25 years, Prosser has experienced at least two revolutions – and expects more in the future. The first revolution was caused by the emergence of new sequencing technology in the early 1990s, which made it possible to analyse hundreds of thousands of species rather than just a few dozen, without the need to grow organisms in the lab. Until this technological breakthrough, microbiologists had studied the soil by characterising the bacteria or "isolates" they could grow in the laboratory, but sequencing eliminates the requirement for lab growth and greatly increases the speed, cost and precision of identification. This has led to the discovery of countless new species of bacteria they never knew existed before.
In the laboratory, bacteria were grown under "unnatural" conditions that biased the outcome, so research would focus on what Prosser calls "domesticated bacteria" rather than on the natural community. Prosser compares this to scientists basing their understanding of bird ecology on the study of domesticated birds (e.g., budgies and parrots), then suddenly discovering the vast diversity of birds outside, in nature.
The method used by Prosser to study what lives in the soil involves a technique called PCR, or polymerase chain reaction (see sidebar) – which helps identify the individual species by amplifying genes from DNA extracted directly from environmental samples, rather than relying on bacterial growth in the lab.
"There were some groupings of bacteria we thought we knew a lot about," says Prosser, "and suddenly we discovered vast, unexpected diversity within these groups and new sub-groupings. In addition, we discovered that 40–60% of bacteria in a typical garden soil belonged to high-level groupings that had not been cultivated in the lab before. There was much more out there we'd simply never seen before. For the first time, we could study these hard-to-grow bacteria."
According to Prosser, "until the 1980s, all our knowledge had been based on what we had grown in the lab," but now researchers attempt to sequence all the genes in a sample to profile all the individual 'species' – like identifying millions of products by reading their barcodes.
Thanks to these new methods, Prosser and his colleagues have discovered more diversity than anyone has seen before – instead of finding 20 or 30 species (a typical result when the bacteria were grown in the lab), they have discovered hundreds of thousands of species, all different. "All our theories until then were based on our knowledge of domesticated species," says Prosser.
Enter the archaea
The second revolution for Prosser was the discovery that archaea took part in the nitrification process and were therefore much more important than scientists used to believe – and could provide the key to understanding more about soil than they'd ever expected. Archaea were thought to be "extremophiles" – organisms that like to live in environments that seem particularly harsh to us, such as thermal springs or very low-pH (acidic) soils, or even environments without any oxygen. In evolutionary terms, plants and animals, including humans, are now thought to be a group within these archaea.
For many decades, microbiologists happily studied bacteria and divided the world into three main domains: plants, animals and bacteria. In the 1980s, however, this theory was turned upside down, with the discovery of a new class of microbes called archaea, while plants and animals were placed in a single group, the eukaryotes.
Prior to this, archaea were regarded as a sub-set of bacteria, but we now know that they make up 4–5% of the micro-organisms present in soil and 40% of the microbes in oceans. New evidence also means that archaea are now defined as a domain of their own, with bacteria defined as the second domain.
Despite the way different domains are defined, every living organism shares some genes, including ribosomal RNA. And Prosser and his scientific colleagues have finally been able to understand what's really happening under our feet by using regions of DNA, the ribosomal rRNA gene, to fingerprint species – and discover the important role played by archaea in soil nitrification. Archaea are hard to grow in the lab, but sequencing has revealed what was "invisible" before.
When archaea started to take centre stage, thanks to these innovative sequencing methods, some scientists found their world turned upside down, but Prosser and his colleagues have embraced the new discovery – and gone on to make further discoveries.
The wonders of nitrification
"We study the specific micro-organisms that control nitrification," says Prosser. "One of our aims is to understand how they break down ammonia-based fertilisers in the soil, forming nitrite which in turn is converted to nitrate, which is then lost (often more than 50%) from the soil by leaching. This reduces the efficiency of fertilisers, added to promote growth of crops, and also creates by-products that lead to air (nitrous oxide) and water (nitrate) pollution – the price paid for our fast-expanding appetite for food. If farmers could control the process better, then crops would be healthier and the environment would not be damaged so much. Current estimates suggest that inefficient use of fertiliser leads to "tens of billions of dollars" of losses worldwide, and ammonia-based fertilisers are increasingly used, with China fast becoming the biggest consumer.
When some ammonia-based fertilisers are added to some soils, says Prosser, nitrification starts almost immediately – these soils are dominated by specific types of ammonia oxidisers that tolerate high concentrations of ammonia. In other cases, nitrification may be delayed for more than a week, because these soils are dominated by ammonia oxidisers that are sensitive to high ammonia. These delays keep ammonia in the soil for longer, keeping it available for crop growth and increasing fertiliser use efficiency, but when the process is faster, ammonia is converted into nitrates that are washed away when it rains, reducing efficiency, or converted by other microorganisms to nitrogen or nitrous oxide, causing atmospheric pollution. These findings have required the use of molecular techniques, and sequencing of nitrifier genes, to characterise the nitrifier communities in the soil and predict the local conditions and communities that will influence fertiliser losses. "We've been able to show the links between the groupings of bacteria, the physiology of the microorganisms and rates of fertiliser loss," says Prosser.
One of the topics that Prosser has focused on in recent years is "the paradox of nitrification in acid soils." This is when nitrification occurs in soil which is particularly acidic, even though the bacteria that are isolated from such soils are not able to thrive in the laboratory under acidic conditions.
This paradox has puzzled microbiologists for many years, but Prosser and his team have been able to solve it, partly thanks to using modern sequencing methods which arose through another revolution in nitrification research. In the past, it was believed that all ammonia oxidisers were bacteria, but about 12 years ago an ammonia oxidiser was isolated that was found to belong to the archaea. Prosser's group targeted a functional gene (a gene that enables an organism to perform a specific function) that is required by all ammonia oxidisers. The sequence of this functional gene is different in bacteria and archaea, making it possible to use molecular techniques to distinguish communities of these two groups of ammonia oxidiser in the soil. But when they analysed the ammonia oxidisers in acidic soil, they discovered something quite unexpected – it was archaea, not bacteria, that dominated nitrification.
Prosser compares the discovery of archaeal ammonia oxidisers to ecologists studying birds (bacteria) and thinking that they know everything about all the organisms that can fly, then discovering insects (archaea). Quite simply, the archaea thrive in low-pH conditions and are good at oxidising the ammonia under these conditions.
To confirm these results, the researchers were able to investigate soils that had been kept at a range of pHs from reasonably acidic (pH 4.5) to slightly alkaline (pH 7.5) for the past five decades at the nearby SRUC (Scotland's Rural University College) site, and were able to isolate an archaeal ammonia oxidiser that was able to grow in the lab under acidic conditions. They also found that sequences of the archaeal ammonia oxidiser functional gene in this organism were distributed in acidic soils all over the world, and were not just a strange phenomenon in Scottish soil. "These archaea therefore provide at least one solution to the paradox of nitrification in acid soils," says Prosser, "but we must remember the surprises that we've had in the past and should not assume that this is the only solution."
Another important finding, with relevance to agriculture and crop growth, emerged from recent research on the response of archaeal and bacterial ammonia oxidisers to different types of fertiliser. There is increasing evidence that archaea dominate when ammonia is released slowly, from slow-release fertilisers or from natural soil processes, while bacteria dominate when high concentrations of inorganic fertiliser are added. In other words, one size does not fit all, and the type of fertiliser that should be used depends on which ammonia oxidisers are present in the soil. This suggests that the type of fertiliser used should be matched with the soil nitrifier community, both to benefit crop growth and to reduce nitrous oxide production, requiring more information on local soil microbial communities.
"In general, science progresses fastest when it is driven by important scientific questions aimed at understanding natural phenomena," Prosser continues, "because questions encourage us to look for answers, rather than trying to fit answers to data after the data have been obtained, and after they have already biased our thinking."
Prosser also quotes the scientist who said, "The problem with molecular biology is that it eliminates the need for thought." In other words, it's easy to get sucked into "stamp collecting" – sequencing genes for its own sake and surveying communities as an end in itself – rather than thinking about which questions are important, what are the important gaps in understanding and what are the mechanisms that drive the interactions between microbes and their environment. There are still many phenomena that soil microbial ecologists cannot explain, but the next time you sit down to dinner, remember that bacteria and archaea are much more important than the chef who prepared it.
Gene genius
The polymerase chain reaction (PCR) is a technique used throughout molecular biology to amplify a single copy of a segment of DNA (deoxyribonucleic acid) across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
To study the microbial diversity of soil, researchers first extract and purify 'total' DNA from the soil. Using PCR, they then amplify a gene called 16S ribosomal RNA that is found in all bacteria and archaea (or 18S ribosomal RNA that is found in plants and animals). To target the group of organisms they are interested in, they use specific primers – two short pieces of DNA – that match sequences at the beginning and end of a region of this gene and that are found only in the target group. Enzymes then make thousands or millions of copies, and the copies are sequenced to identify the different members of the target group present in the sample.
Recent advances in the sequencing of environmental DNA and RNA have significantly decreased sequencing costs and enable rapid analysis of soil microbial communities and the assembly of "population genomes" from soil DNA, providing more and more information on the potential of the organisms present to perform different functions.
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"Soil Bacteria". Science Scotland (Issue Twenty-one)