The Diversity of Dirt What is the role of the soil microbial community in ecosystem functioning? Dr Martina Girvan June 2012 Talk Structure •How Diverse is Soil? Back to the Classroom! •Exploitation of Genetic Diversity and how if relates to Functional Diversity •Research Case Study •Why it matters June 2012 Common View of Biodiversity Soil Microbial Diversity and Function July 9, 2012 Page 3 Traditional Taxonomists View of Biodiversity • “the biological heterogeneity of a system, it encompasses species richness and distribution” • Animalia Eukaryota • Plantae • Fungi • Protista Prokaryota • Monera Soil Microbial Diversity and Function July 9, 2012 Page 4 Current Taxonomic View of Biodiversity • Animalia • Plantae • Chromista • Fungi • Protozoa • Archaezoa etc. Bacteria • Bacteria (many kingdoms) Archaea • Archaebacteria (many kingdoms) Soil Microbial Diversity and Function July 9, 2012 Page 5 Bacteria Archaea Methanosarcina Proteobacteria Cyanobacteria Gram positives Slime moulds Entamoebae Thermoproteus Green Non-sulphur Bacteria July 9, 2012 Animals Plants Ciliates Flagellates Trichomonads Microsporidia Diplomonads LUCA Soil Microbial Diversity and Function Fungi Methanococcus Pyrodictium Thermatogales Halophiles Methanobacterium Thermococcus Flavobacteria Eucarya Page 6 Different Phenotypes Same Metabolic Pathway/ Same Phenotype Different Metabolic Pathway Methanocella paludicola Methanogenesis: 4H2 + CO2 → CH4 + 2H2O Nitrosotalea devanaterra Autotrophic Ammonia oxidation: NH3 + O2 NO2- + H2O CO2 organic C Desulfopila aestuarii Sulphate reduction with acetate: Oxygen-respiration with glucose: C6H12O6 + 6O2 → 6CO2 + 6H2O Soil Microbial Diversity and Function July 9, 2012 CH3COO- + SO42- → 2CO2 + HS- + 2H2O Page 7 Soil Diversity • Eukaryotic diversity on Earth was estimated to be approx. 8.7 million (8.7 x 106) species in total1 • One individual sample of 10g soil was estimated to contain 8.3 million (8.3 x 106) microbial species2 Soil Microbial Diversity and Function July 9, 2012 Page 8 Why Such Diversity?: Heterogeneity and spatial structure of soil microbial communities Variability in physicochemical properties e.g. water content, oxygen availability and pH high Aerobes (e.g. pseudomonads) O2 availability high pH low low Soil Microbial Diversity and Function July 9, 2012 Anaerobes (e.g. fermentative bacteria (organic acids) methanogenic archaea (methane)) Page 9 Why Such Diversity?: Heterogeneity and spatial structure of soil microbial communities Variability in soil structure e.g. avoidance of predation Fast-growing organisms Protozoa Slow-growing organisms Soil Microbial Diversity and Function July 9, 2012 Page 10 Why Such Diversity?: Heterogeneity and spatial structure of soil microbial communities Variability in carbon sources e.g. root exudates Plant root Low molecular weight compounds - Sugars - Amino acids - Carboxylic acids Soil Microbial Diversity and Function July 9, 2012 Root exudates Page 11 hy Such Diversity?: Heterogeneity and spatial structure of soil microbial communities Variability in energy sources e.g. light Phototrophs (e.g. cyanobacteria) Chemoheterotrophs (e.g. Bacillus sp. Pseudomonas sp.) Soil Microbial Diversity and Function July 9, 2012 Page 12 Soil Microbial Diversity Summary • Bacterial and Archaea are more different from each other than we are from nematodes or indeed Bacteria, both in taxonomic and functional terms • Soil represents a huge range of different habitats for soil microbes • Different microbes have evolved to take advantage of specific ecological niches • Therefore, thousands of habitats = thousands of species Soil Microbial Diversity and Function July 9, 2012 Page 13 Talk Structure •How Diverse? •Exploitation of Genetic Diversity and how if relates to Functional Diversity •Research Case Study •Why it matters June 2012 Bio-prospecting for Genetic Resources 5 Please let the Pseudomonads be ready! 3 Soil Microbial Diversity and Function July 9, 2012 4 5 Page 15 Functional Resources • Mineralisation of Organic and Inorganic Substrates – E.g. Leaf litter decomposition, sulphur oxidation and reduction, ammonification, nitrogen fixation, methanogenesis and methane oxidation – Bacteria and Archaea exploit inorganic compounds for energy and so complete chemical pathways , therefore major biogeochemical cycles such as carbon, nitrogen, sulphur, phosphorus are inextricably linked to soil microbes • Water Purification – Reedbed sewage purification and other mainstream sewage treatment plants by aerobic digestion • Carbon Sequestration – Peatbogs only 3% land surface but contain nearly a third of the worlds soil organic carbon6 Soil Microbial Diversity and Function July 9, 2012 Page 16 Biodiversity of DNA and Function Combine to Create Functional Soil Ecosystems • Genetic diversity within and between species – Provide novel genes and the ability to adapt • Functional diversity – Different species with different genetic components may provide similar functions within an Ecosystem • Ecosystem – It is the functional elements and interrelation of these species which comprise an Ecosystem Soil Microbial Diversity and Function July 9, 2012 Page 17 Why unfamiliar to mainstream ecology? • Not visible • Not accessible to natural historians • 99.9% of bacteria cannot be cultured so not accessible to traditional microbiologists • Advances in study became possible due to complex DNA amplification techniques, Metagenomics • Difficult and expensive (also just a tad unglamorous, NOT like CSI or the X Files) Soil Microbial Diversity and Function July 9, 2012 Page 18 Talk Structure •How Diverse? •Exploitation of Genetic Diversity and how if relates to Functional Diversity •Research Case Study •Why it matters Soil Genetic Diversity7 soil DNA extraction insitu/ cell separation Calculate diversity of community profile mechanical cell lysis buffer variations DNA purification chemical cell lysis humic inhibitors sephedex columns caesium chloride gradient agarose gel purification PEG/ethanol precipitation PCR functional gene 16/18S DNA full length sequence Partial sequence clone analysis fragment length heterogeneity DGGE analysis more diverse Soil Microbial Diversity and Function July 9, 2012 Page 20 less diverse Different Measurable Components of Soil DNA RNA Culturable Present Active Traditionally Visible Soil Microbial Diversity and Function July 9, 2012 Page 21 Microcosm experiment with copper and benzene applied at time 0 control copper benzene 16S rDNA analysis Analysis of 16S rDNA sequences showed the diversity of both the copper and benzene contaminated communities to be significantly reduced by week 9 weeks 0 1 4 9 0 1 4 9 0 1 4 9 Shannon index of diversity 3.5 Soil Microbial Diversity and Function 3 control copper 2.5 benzene 2 1.5 July 9, 2012 week 0 week 1 Page 22 week 4 week 9 Microcosm experiment with copper and benzene applied at time 0 16S rRNA analysis control copper benzene Analysis of 16S rRNA sequences again showed a significant reduction in diversity of both the copper and benzene contaminated communities by week 9 Shannon index of diversity weeks 0 Soil Microbial Diversity and Function 1 4 9 0 1 4 9 0 1 4 9 3.5 3 control copper 2.5 benzene 2 1.5 July 9, 2012 week 0 week 1 Page 23 week 4 week 9 14C mineralisation assays showing functional community capacity Wheat shoot (broad niche) mineralisation measured by 14C 0.25 % Mineralisation Although genetic diversity was significantly reduced in benzene and copper contaminated soil by week 9 wheat shoot mineralisation was unaffected 0.30 0.20 0.15 0.10 0.05 control copper benzene 0 18 2,4, DCP (narrow niche) mineralisation measured by 14C week 1 week 4 week 9 week 1 week 4 week 9 However 2,4 DCP mineralisation was severely inhibited until recovery by week 9 % Mineralisation 1.5 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 Soil Microbial Diversity and Function July 9, 2012 Page 24 Summary of Research Findings • Soil is genetically resistant • Soil also shows functional resilience • Some functional redundancy for broad scale niche utilisation • BUT narrow niche function was impaired • Overall genetic diversity confers the ability for soil to recover functionally • We do not know enough about soil processes to risk it! Soil Microbial Diversity and Function July 9, 2012 Page 25 Talk Structure •How Diverse? •Exploitation of Genetic Diversity and how if relates to Functional Diversity •Research Case Study •Why it matters June 2012 Summary • • • • • • Bacterial and Archaeal Soil microbial diversity is vast equalling the whole of the Eukaryotes in the world in just 10g of soil This diversity is caused by heterogeneity in structure , energy sources and oxygen gradients etc. The functional elements of soil are vital to life on earth but poorly studied due to technical difficulties There is much functional redundancy among microbial populations. Therefore using macro-ecological measurements of diversity may not apply Functional diversity may be more realistic means of measuring the “health” of microbial communities Although molecular techniques are important we can only achieve a comprehensive analysis of functional diversity if we increase the culturability of soil microorganisms Soil Microbial Diversity and Function July 9, 2012 Page 27 Have We Got It Right? Us of Herbivores Pe rce p tio n Invertebrates Plants Invertebrates Soil Soil Microbial Diversity and Function July 9, 2012 Page 28 ty? ali Re im po rta nc e Carnivores Tom Curtis8 (2006) Microbial ecologists: it's time to 'go large' Nature Reviews Microbiology 4, 488 “For if the last blue whale choked to death on the last panda, it would be disastrous but not the end of the world. But if we accidentally poisoned the last two species of ammonia oxidisers, that would be another matter.” Soil Microbial Diversity and Function July 9, 2012 Page 29 References 1. Mora C, Tittensor DP, Adl S, Simpson AGB, Worm B (2011) How Many Species Are There on Earth and in the Ocean? PLoS Biol 9(8): e1001127. doi:10.1371/journal.pbio.1001127 2. Gans J, Murray Wolinsky, and John Dunbar (2005) Computational Improvements Reveal Great Bacterial Diversity and High Metal Toxicity in Soil Science : 309 (5739), 1387-1390. 3. Rolf Daniel (2005) The metagenomics of soil Nature Reviews Microbiology 3, 470-478 4. Knowles, B. H. & Ellar, D. J. (1988) Differential specificity of 2 insecticidal toxins from Bacillus thurinigensis var aizawai. Molecular Microbiology, 2, 153-157. 5. Meikle, A., Killham, K., Prosser, J. I. & Glover, L. A. (1992) Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil. FEMS Microbiology Letters, 99(2-3), 217-220. 6. Koehler, A K, Sottocornola, M And Kiely, G. Global Change Biology (2011) 17: 309–319 7. Girvan M. S., L.A. Glover, K. Killham, C. Campbell, J.I. Prosser. 2005. Bacterial Diversity Promotes Community Stability and Functional Resilience after Perturbation. Environmental Microbiology. 7: 301–313 Soil Microbial Diversity and Function July 9, 2012 Page 30 Thank You for Listening Thanks to Dr Graeme Nicol of Aberdeen University for all his help and advice, a great lab partner Thanks to the AECOM ecology team for listening! Please do contact me for further information [email protected] June 2012
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