Developments in Soil Microbiology Since the Mid 1960S
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Geoderma 100Ž. 2001 389–402 www.elsevier.nlrlocatergeoderma Developments in soil microbiology since the mid 1960s Heribert Insam) Institute of Microbiology, UniÕersity of Innsbruck, Technikerstrabe 25, A-6020 Innsbruck, Austria Received 10 January 2001; received in revised form 19 February 2001; accepted 22 February 2001 Abstract Since the 1960s, soil microbiology underwent major changes in methods and approaches and this review focuses on the developments in some selected aspects of soil microbiology. Research in cell numbers of specific bacterial and fungal groups was replaced by a focus on biochemical processes including soil enzyme activities, and flux measurements of carbon and nutrients. Ecologists focused on soil microbial pools whereas soil microbial biomass as an important source and sink of nutrients were recognized in agriculture. Soil microbiologists started to use structural components like phospholipid fatty acids for quantification of specific microbial groups without the need to cultivate them. In the last decade, molecular approaches allowed new insights through the analysis of soil extract DNA showing an unexpected diversity of genomes in soil. At the end of the review a brief outlook is given on the future of soil microbiology which ranges from in situ identification of bacteria, to routine assays of microbial communities by microarray technology. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Soil microbiology; Review; Enzymes; Microbial biomass; N turnover; Molecular ecology 1. Introduction Microbial ecology, and more specific, soil microbiology is a dynamic and growing subdiscipline of soil science. There are a number of new journals in this field but also traditional soil science journals increasingly attract soil microbiological papers. In 1967 the first issue of Geoderma was published. Thirty-four years is a long time span in most scientific fields and during these years soil microbiology has developed from a playground of soil scientists, microbiologists and soil chemists to an own subdiscipline. This review on soil microbiology on the occasion of 100 volumes of ) Tel.: q43-512-507-6009; fax: q43-512-507-2928. E-mail address: [email protected]Ž. H. Insam . 0016-7061r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved. PII: S0016-7061Ž. 01 00029-5 390 H. InsamrGeoderma 100() 2001 389–402 Geoderma aims to introduce to soil scientists the importance of microbiological aspects of the soil and the new insight offered by new techniques. It gives a brief historical overview of the developments within soil microbiology, and some thoughts on future prospects. 2. Driven by methods Soil is very complex with diverse niches offered to soil microorganisms. Having to deal with solid, liquid and gaseous phases, soil microbiologists were in need of suitable methods for studying their subject since the beginnings of this field. ‘Ecology of soil microorganisms’Ž. Parkinson et al., 1971 was the standard reference for soil microbiolo- gists 30 years ago and it was a relatively thin booklet with 116 pages. Nowadays, methods books in soil microbiology are abound and they usually exceed 500 pagesŽ e.g., Weaver et al., 1994; Alef and Nannipieri, 1995; Schinner et al., 1996; Van Elsas et al., 1997; Hurst et al., 1997.Ž. New windows into the black box are being opened Fig. 1 . In soil microbiology it was realised that simply extracting soils and counting microorganisms is not enough to characterise the soil microbiota and its significance for the functioning of soilsŽ. e.g., Macura, 1974 . Prevalent methods of cell enumeration account for a very small fraction of the total number of microbes, and reveal no information about the activity of the counted organism. Both methods of extraction and enumeration are subject to bias and differences may be as large as three orders of Fig. 1. The soil as a Black Box. Soil microbiologists open up new windows to investigate soil microbial communities, their composition and functionsŽ CLPP, Community level physiological profiles; PLFA, Phospholipid fatty acids; details see text. H. InsamrGeoderma 100() 2001 389–402 391 Table 1 Ž.a Effect of extraction method on total microbial counts Ž Smith and Stribley, 1994 . Method of extraction Bacterial numbers per gram inorganic matter Dispersed soilŽ. control 8.5=108 Repeated centrifugation and resuspension 1.5=109 Ž.Hopkins et al., 1991 Aqueous two-phase partitioning 1.3=1010 Ž.Smith and Stribley, 1994 Ž.b Total, viable, and culturable soil bacteria of a barley field soil Ž Winding et al., 1994 .Ž simple extraction. Method of counting Bacterial numbers gy1 soil Acridine orange direct countŽ.Ž. AODC total bacteria 1=109 CTC reducing bacteriaŽ. metabolically active bacteria 3.5=107 Microcolony-forming unitsŽ. micro-CFU 2.5=107 Žbacteria that are able to perform a few, but not more cell divisions on agar. Ž.viable but not culturable bacteria a Colony-forming unitsŽ. CFU on agar plates 7=106 Ž.culturable bacteria a a Longer incubations, up to 64 days, increased the numbers of CFUs. magnitudeŽ. Table 1 . Despite this shortcoming, cell enumeration methods have long been the prime choice for soil microbiologists to obtain quantitative data. Counting methods do have virtue in cases when a specific organism is studied but it is advisable to crosscheck the figures on microbial numbers in relation to the applicability of the analytical method. The data in Table 1 show that with traditional cultivation methods on agar plates only a small percentage of the total microflora is accounted for, and for the remaining 99% physiologic and taxonomic information is lacking. For most soil microbiologists such data were not satisfactory for understanding soil functioning and the consequence was to attempt to measure processes like enzyme activities or N fixation and nitrification, or more unspecific processes like CO2 evolution or heat generation. 3. Soil enzymes Enzymes in soil may be extra- or intracellular. Extracellular enzymes are necessary for the breakdown of organic macromolecules, like cellulose, hemicelluloses or lignin whereas intracellular enzymes are responsible for the breakdown of smaller molecules like sugars or amino acids. Soil enzymes are predominantly of microbial origin and are closely related to microbial abundance andror activity. Biochemical tools that allowed rapid measurement of soil enzyme activities made soil enzymology fashionable in the late 1960s, and such tools remained widely used for 20 years. Numerous enzymes have been tested on their suitability for soil investigations; the choice of method will largely depend on the geochemical cycleŽ. e.g., C, N, P or S under investigation. Using 392 H. InsamrGeoderma 100() 2001 389–402 databases from the Institute of Scientific InformationŽ. ISI it has been attempted to quantify which enzyme tests are most frequently used today and these are urease Ž.Ž.Tabatabai and Bremner, 1972 , phosphatase Hoffmann, 1967 and dehydrogenase Ž.Trevors, 1984 , indicating the acceptance of the methods in the scientific community. A major weakness of enzyme tests is that the actual microbial activity of a soil is not well reflected. Moreover, the tests show ‘historic’ features of enzymes bound to soil organic matter or clay minerals. Therefore, Visser and ParkinsonŽ. 1992 disputed the suitability of enzyme assays for microbial activity and soil quality assessments, with the exception of dehydrogenase because its biological properties make it unlikely to be present in soil in an extracellular stateŽ. Skujins, 1978 . To overcome the interpretation problems of single enzyme tests, BeckŽ. 1984 proposed a soil microbiological index calculated from microbial biomass, reductase and hydrolase activities. This index, however, never became popular. In another attempt to propose such an index, Trasar-Cepeda et al.Ž. 1998 found a very close relation of total N with a linear combination of soil microbial biomass C, mineralised N, phosphomo- noesterase, b-glucosidase and urease activity. This test set was proposed as a soil quality index closely related to soil sustainability. The problem with both Beck’sŽ. 1984 and Trasar-Cepeda et al.’sŽ. 1998 approaches is that the usefulness of the enzyme tests used as components of their indices is disputed, and universally accepted enzyme tests are still lacking. The use of fluorogenic substrates has recently been proposed for studying microbial activities. Miller et al.Ž. 1998 used 4-methylumbelliferyl N-acetyl-b-D-glucosaminide, that, when hydrolysed, releases 4-methylumbelliferone which fluoresces and can be detected in nanomolar concentrations. This test specifically determines fungal chiti- nolytic activities. Assays using labelled substrates directly test for substrate degradation and may be performed in microplates, they need smaller sample sizes than traditional enzyme tests and allow measurement of many parallels in one assay. Community-level physiological profilingŽ. CLPP , which is another type of ‘enzyme assay’, has been proposed by Garland and MillsŽ. 1991 . With this method, the ability of microbial communities to degrade a set of up to 95 different substrates is tested in one single assay. A redox indicator in the Biolog w microtiter plates indicates if the specific substrate is used as an energy source. CLPPs have been shown to be very sensitive indicators of disturbancesŽ. e.g., Mayr et al., 1999; Yan et al., 2000 and microplates Ž.EcoPlates especially designed for environmental applications Ž Insam, 1997 . are now available. Summarizing,