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Microbial Biomass Measurement Methods. Chapter · January 2004
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MICROBIAL BIOMASS MEASUREMENT METHODS
K.R. Islam, Ph.D Soil and Water Specialist The Ohio State University South Centers Piketon, OH 45661
Tel. 740-289-2071 Fax: 740-289-4591 Email:
[email protected]
S. W. Wright, Ph.D. Horticulturist/Ecologist The Ohio State University South Centers Piketon, OH 45661
Tel. 740-289-2071 Fax: 740-289-4591 Email:
[email protected]
K. R. Islam and S.W. Wright The Ohio State University South Centers Piketon, Ohio, U.S.A. MICROBIAL BIOMASS MEASUREMENT METHODS KEYWORDS: Chloroform fumigation, Microwave irradiation, Substrate-induce respiration, Rehydration, Freeze-drying, ATP extraction, Phospholipids, Extraction, Incubation, Extracted C
INTRODUCTION Soil microbial biomass (SMB) is an active component of the terrestrial ecosystem, which regulates many critical functions and properties related to soil and environmental qualities. The functions and processes include source-sink in nutrient cycling, decomposition of organic residues, structural stability, and indicator of soil pollution and bioremediation (1, 2, 3). No standard method for measuring SMB exists, but several widely differing approaches have been developed over the last two decades. Methods used for measuring SMB are briefly discussed.
I.
DIRECT METHODS TO MEASURE SOIL MICROBIAL BIOMASS In direct methods, microorganisms are determined by colony forming units counted on soil
dilution series using most probable number (MPN) and/or by direct microscopic counting methods. In MPN method, soil samples are dispersed in a series of dilution to estimate population density based on the presence or absence of microbial cells (4). Thus, if microbial growth is observed in the 10-4 but not in the 10-5 dilution, the number of cells is estimated in between 104 - 105. The direct microscopic method involves dispersing a known amount of fresh soil in a known volume of water or dilute agar media is smeared over a known area on a glass slide to count for
microbial cells using fluorochromes. The SMB measurements from cell dry weight and volume characteristics (5) as follows: Bacterial biomass (µg/g oven-dry soil) = NV Bb 106 Where N is the number of bacteria/g of oven-dry soil, V is the average volume of bacterial cell (µm3), Bb is the biomass/volume conversion factor (0.22 to 0.33 x 10-12 g/µm3). Fungal biomass (µg/g oven-dry soil) = L Πr2 Bf 106 Where L is the mycelia length in µm/g oven-dry soil, r is the average radius of mycelia (µm), Bf is the biomass/volume conversion factor (0.2 to 0.33 x 10-12 g/µm3). Soil MPN method based on dilution is widely applicable to bacterial cell counting but for fungi it is only valuable for spore or other propagule counting. The direct microscopic is a tedious procedure, and often yields result 10 to 100 times greater than the soil dilution techniques.
II.
INDIRECT METHODS TO MEASURE SOIL MICROBIAL BIOMASS In indirect methods, the SMB is assessed using biochemical, chemical and physical
principles for determination of a particular cell constituent such as C, N, P, S, ATP, and phospholipids of microbes (6, 7, 8, 9, 10, 1, 12, 13, 14).
a)
Chloroform (CHCl3) Fumigation Incubation and Extraction Methods The CHCl3 fumigation is a reference method to determine SMB since it was first developed
(9). The rationale for the CHCl3 fumigation incubation (CFI) method is that the fumigant lysed the soil microbes and the resulting increase in CO2 evolution from fumigated soil as compared to unfumigated soil over a 10-d incubation period at 25oC is directly proportional to the amount of C in
the SMB (9). The amount of CO2 released is determined by absorbing in 0.5M NaOH followed by acid-base titration to calculate the SMB. SMB = Fc/Kc Where Fc is the net flush of CO2 from fumigated and unfumigated soils, respectively during incubation, and Kc is a coefficient of 0.45 (9). In CHCl3 fumigation extraction (CFE) method, the post-fumigated soil is extracted with suitable extractants for flush of C, N, P and S compared to unfumigated soils (6). Soil extracts were analyzed for C, ninhydrin-reactive-N (NRN), P and S to calculate SMB (6, 13). SMB = 2.68V – 44.1 (6) Where V is the net flush of C from fumigated and unfumigated soils, respectively extracted by neutral 0.5M K2SO4. SMB = 20.0 x NRN if soil pH is >5.0 (13)
SMB = 35.3 x NRN if soil pH is
