Soil Biology & Biochemistry 43 (2011) 1619e1620
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Citation classics
Soil Biology & Biochemistry Citation Classic X Richard G. Burns* School of Agriculture and Food Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia
Fatty acid research pervades numerous processes in the industrial world. It has relevance to biofuels, surfactants, soaps, paints, plastics, cosmetics, insecticides and disinfectants and it is hard to escape the hoopla associated with polyunsaturates and the many foods and dietary supplements containing u-3 and u-6 fatty acids. The focus of this latest SBB Citation Classic article is more prosaic and receives somewhat less media attention - even though interrogating the soil microbial community is of great and increasing significance to our understanding and stewardship of the environment. This is because the composition and activities of the soil microbial world govern elemental cycles and the development and retention of soil fertility e processes that are under the onslaught of climate change and may not prove as robust and resilient as once believed. Phospholipids fatty acids (PLFA) are essential structural components of the cell membranes of all living cells. The lipid bilayer has hydrophilic outer surfaces and an hydrophobic inner component, a structure and configuration that gives it a number of functions - in addition to the well known ones of mechanical support and cellular integrity. These include regulating the uptake and secretion of ions and molecules (often via trans-membrane proteins), electron transport, controlling pH, contributing to adhesion and biofilm formation, playing a part in the communications between microbes and microbes and microbes and plants (including fungal-plant pathogen interactions) and even serving as an energy source during stress and starvation. PLFAs are synthesized during microbial growth and, in general, rapidly decompose after cell death. Therefore, the concentration of total PLFAs is a measure of the microbial biomass and the individual fatty acids provide details about community structure. PLFAs are usually extracted from soil by chloroform/methanol/water, separated from non-polar lipids using silicic acid columns, detached from their glycerol backbone, and then esterified prior to gas chromatographic analysis. The resulting lipid profile is compared to a data base. However, this provides only modest phylogenetic resolution, because it relies on an understocked library of phospholipid profiles from previously cultured microorganisms. Organic chemistry 101 tells us that fatty acids are composed of carbon chains that are either mono- or poly unsaturated or fully saturated. For the unsaturated ones the shorthand used here, and in
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most soil biology papers, refers to the number of carbon atoms and the number of double bond(s) and their position counted from the methyl group (u) end. For example, in 16:1u5, 16 refers to the number of carbon atoms, 1 the number of double bonds and 5 the location of the double bond in relation to the number of C atoms from the terminal methyl group. When used, the suffixes c (cis) and t (trans) refer to isomers. The saturated fatty acids are described similarly except that additional information is provided by various prefixes: the location of the cyclopropyl (cy) ring and the presence of a methyl (Me) group either on the penultimate carbon counting from the u end (i, iso) or on the antepenultimate carbon atom three from the u end (a, anteiso). Monounsaturated fatty acids commonly used for microbial identification are: 16:1u5 (bacteria, arbuscular mycorrhizal fungi), 16:1u7c (bacteria), 16:1u8 and 18:1u8 (methanotrophs), 18:1u7c (bacteria) and 18:1u9 (fungi). Microbial polyunsaturated fatty acids include 18:2u6,9, 18:3u3,9,12 and 18:3u6,9,12 which are all typical of fungal membranes. The saturated fatty acids are long-chain carboxylic acids that usually have between 12 and 24 carbon atoms and no double bonds. They include the branched i15:0, a15:0, i16.0, i17.0 and a17:0, which are all Gram-positive biomarkers, and the cyclopropyl saturated ones, cy17:0 and cy19:0, which are considered to be indicators of Gram-negative bacteria. The methyl branched fatty acids, 10Me16:0, 10Me17:0, 10Me18:0 and 10Me19:0 are diagnostic for the actinobacteria. There is disagreement, some outright contradictions (as well as many proof reading errors) in the literature, but microbial biomass and the ratios of bacteria to fungi are often but not exclusively assessed using some or all of the following PLFAs: Gram-positive bacteria: a15:0, i15:0, a16:0, i16:0, a17:0 and i17:0; Gramnegative bacteria: 16:1u7c, 16:1u9, 18:1u5c, 18:1u7c, cy17:0, cy19:0; and total bacteria all these plus 15:0, 17:0, 16:1u5, 17:1u6, 17:1u7 and those with methyl groups. Fungal biomass is often correlated with 18:2u6,9 and sometimes 18:1u9c. The overwhelming ambition of many soil biologists is to identify microbial genera and species and assess diversity and function: this sometimes means turning a blind eye to the inherent limitations of all the so-called signature molecules. The handwriting is blurred at best and deciphering the text can be a real challenge. This is because, even though more than one hundred PLFAs have been extracted from soils few, if any, are unique to a particular genotype; in the fungal world (1.5 million species and counting) there are only ten different fatty acids. Furthermore, the presence and concentration of fatty acids can
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change according to growth phase, carbon and nitrogen sources, oxygen, pH, and a host of stress factors. For example, during starvation, cells shrink and the some fatty acids, such as 16:1u7c, are metabolised whilst others, including 16:1u7t, increase: nutrient concentrations influence the relative proportions of fatty acids. In fact, the 17:0cy to 16:1w7c ratio is sometimes used as a measure of nutritional stress. Additionally, some fatty acids (e.g. 18:1w9, 18:2w6,9 and 18:3w3,6) are found in plant roots, algae, cyanobacteria and even nematodes and this potential ’contamination’ must be taken into account when preparing soil for extraction. Then there are the increasingly important archaea with membranes composed of ether lipids rather than ester lipids: fatty acids that are not extracted by the usual procedures. Of course, underpinning all this interpretive uncertainty are the many factors which determine the presence and expression of fatty acid synthases. The three authors of this latest Citation Classic, Åsa Frostegård, Anders Tunlid and Erland Bååth, are past masters in the use and interpretation of PLFA profiles. They re-visit their much-cited paper (Frostegård et al., 1993), offer an honest reappraisal of the technique, remind us of the advantages and disadvantages, and alert us to the frequent misinterpretations. Their research illustrates how the extraction and analysis of phospholipid fatty acids from microbial cell membranes has shone a bright light on our understanding of microbial communities and their functions in soil. Åsa Frostegård conducted her PhD research at Lund University in Sweden and it was this that set her on the path to pre-eminence in the field of microbial PFLA analysis and interpretation. She monitored changes in membrane fatty acids when soil was subjected to a range of stresses including pH and heavy metal contamination. Following this, she took up a post-doctoral fellowship in France working with Pascal Simonet on the then (and now) hot topic of horizontal gene transfer in soil. In 1996 Åsa returned to Lund to further progress her studies of soil molecular ecology. Then, in 2000, she was appointed to a professorship at the Norwegian University of Life Science in Aas, just a short drive south of Oslo, where she continued her successful collaboration with Lars Bakken. Together they have co-authored eleven papers. In the past decade, Professor Frostegard’s research team has grown in size and she has published a number highly influential papers (ten have been cited more than 150 times) on aspects of the nitrogen cycle including the diversity and phylogeny of rhizobia as well as the regulation of denitrification and the control of nitrous oxide reductase and consequent N2O emission (http://www.umb.no/nitrogengroup). Åsa’s imaginative and expert combination of molecular and chemical approaches to soil microbiology puts her at the forefront of research in this area. When not pushing back the boundaries of our understanding of soil biology, she is reading, keeping fit, taking care of her 11-year old daughter and developing her already significant culinary skills. Anders Tunlid has spent much of his working life at Lund University where he has been Professor of Microbial Ecology for the past twelve years. A critical early period in his career, especially as far as this Citation Classic is concerned, was a three year post-doc at the University of Tennessee in Knoxville working with David White. David was a pioneer (and sometimes a lone proponent) in the use of microbial fatty acids in microbial ecology and started publishing papers on the subject in the mid-1960s. Anders took his already
honed GC and MS skills to the USA and worked with David White during this key period. In more recent times, Prof. Tunlid’s research has focussed on many aspects of microbial ecology and fungal genetics and his interests extend far beyond lipid biomarkers. His publications on nematode-trapping and mycorrhizal fungi are impactful as are his forays into the complex world of plankton in marine environments. Anders, who is an elected member of The Royal Swedish Academy of Sciences, has a prominent international reputation and is the recipient of numerous invitations to present keynote talks throughout the world. When he can catch his breath he is out on his kayak or spending time in his summer house on the Baltic Sea island of Öland. Erland Bååth is Professor of Microbial Ecology at Lund University and his is a name known to soil biologists throughout the world. He has published in excess of 150 papers (SBB has been the fortunate recipient of 48 of these) and 21 have been cited more than 100 times. Erland is a truly international scientist and has forged productive collaborations with scientists from Scotland, England, France, Italy, Germany, Spain, the Netherlands, Austria, Poland, Lithuania, Sri Lanka, Australia, Norway, Finland, Denmark and Sweden. The list of his co-authors is a who’s who of soil biology: Ruess, Pennanen, Clarholm, Ritz, Bloem, Bakken, Bardgett, Brookes, Witter, Fritze, Fierer, Jones, Olsson, Guggenberger, Söderström, Insam, Griffiths, and many more. Erland’s research has embraced not only the development of techniques for identifying and characterising the soil bacterial and fungal (including ecto- and arbuscular mycorrhiza) communities but also methods to estimate microbial growth rates in soil and the disruptions imposed by tillage, fire, fertilizers, antibiotics, acid rain, metals, pesticides and so on. He is a long standing editorial board member of Soil Biology & Biochemistry. When not being an international scientist, Professor Bååth is can be found doing something rather different: he is an enthusiastic chorister adding his sonorous tones to 16th and 17th century madrigal recitals. In the late 1990s PLFA analysis was coupled with stable isotope probing (PLFA-SIP) of 13C/12C ratios and this allowed many processes and fluxes to be evaluated and trophic relationships to be revealed. PLFA-SIP has proved to be a hugely powerful tool in identifying microbial populations in soils and sediments, understanding the impacts of changing land use, shedding light on the complex ecology of methane oxidation and disentangling many animal-plant-microbe interactions. In this last mentioned context, when fatty acids survive transfer between organisms (e.g. bacteria to ciliates to copepods) they provide clues to diet and feeding strategies as well as preyepredator interactions. Recently PLFA-SIP has been combined with 16S ribosomal RNA-SIP analysis to add yet another level of sensitivity which illustrates how tried, tested and reliable chemical analysis can be combined with isotopic and molecular techniques to reveal not only what is going on in soil but also identify the important players. Reference Frostegård, Å, Bååth, E., Tunlid, A., 1993. Shifts in the structure of soil microbial communities in limed forest soils as revealed by phopholipid fatty acid analysis. Soil Biology & Biochemistry 25, 723e730 (Cited 419 times).