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Microbial Biomass Measurement Methods.

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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: islam.27@osu.edu

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: wright.705@osu.edu
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 <5.0 (13)

Both CFI and CEF methods yield good estimates of SMB but CHCl3 is a biohazard. Also

the CFI method is affected by high organic matter content, organic amendments, low pH and soil

waterlogged conditions, and is time consuming and involves several steps (1, 9). The CFE method

is fast and useful where CFI does not work (6). A portion of the SMB may be insensitive to CHCl3

fumigation.

b) Microwave (MW) Irradiation Incubation and Extraction Methods

The underlying principle of the MW irradiation method is to use non-thermal MW energy

@ 800 J/g oven-dried equivalent of field-moist soil in plastic tubes with punctured caps to disrupt

the microbial cells and then incubate or extract the MW and unmicrowaved soils to measure flushes

of C (8). In MW irradiation incubation method, both MW and unmicrowaved soils are incubated for
10-d in the dark at 25oC and the flush of CO2 is absorbed in dilute solution of NaOH followed by an

acid-base titration to calculate the SMB.

SMB = CO2-CMW/KMI.

Where, CO2-CMW is the net flush of CO2 from MW and unmicrowaved soils, respectively, and KMI

is a coefficient of 0.341(8).

In MW irradiation extraction method, the MW soil is extracted for flush of C by neutral

0.5M K2SO4 compared to unmicrowaved soil (8). An automatic analyzer with UV-persulfate

oxidation and IR detection, or rapid colorimetric (8, 15) or titrimetric method is used to determine

extracted C for the SMB measurement.

SMB = CEXTMW/KME.

Where CEXTMW is the net flush of C from MW and unmicrowaved soils, respectively, and KME is the

extraction coefficient (0.213) of the SMB (8).

The MW irradiation method is an alternate approach to CFI and CFE methods for rapid,

precise, safe and reliable measurement of SMB (8). This method is very economical. To avoid

release of non-biomass C from soil, the MW oven has to be calibrated before use.

c) Rehydration Method

Rewetting of air-dry soils with dilute salt solution or water is basis for the rehydration

method for determining SMB (16, 17). Upon rehydration, the desiccated microbial cells in air-dried

soils are disrupted and released intracellular C compounds. The extracted C is analyzed by a rapid

K2Cr2O7 oxidation method to determine the SMB content.

SMB = Ec/0.23

Where Ec is the net flush of 0.25M K2SO4 extracted C from air-dried and field-moist soils,
respectively, and 0.23 is extraction coefficient (17).

The rehydration method is a simple procedure for SMB determination, which does not use

hazardous chemicals but prolonged air-drying of soil often releases non-biomass C. A portion of the

SMB may be insensitive to air-drying.

d) Freeze-dried Soil Extraction Method

The principle of the method is that extraction of freeze-dried soils with either 0.5 M

K2SO4 or 0.5 M NaHCO3 releases cytoplasmic C compounds from desiccated and disrupted

microbial cells (7). The extracted C is analyzed by a rapid colorimetric method (15) to calculate

the SMB contents.

SMB = C- K2SO4/0.152 SMB = C- NaHCO3/0.257

Where C-K2SO4 and C-NaHCO3 are the net differences in C extracted by 0.5M K2SO4 and

NaHCO3 from freeze-dried and field-moist soils, respectively.

This is a precise, reliable and safe method for measuring SMB but it requires trained

personnel and sophisticated equipment.

e) Adenosine Triphosphate Extraction Method

The principle of the method is that by using suitable reagents, soil microbial cells are

disrupted rapidly followed by stabilization of ATP with deactivating synthesis and degradative

enzyme processes, and extraction of ATP from the soil matrix followed by determination using

luciferin-luciferase light reactive system to calculate SMB contents (18, 19).

1 µg ATP = 250 µg of SMB

Since the ATP is degraded rapidly during extraction and adsorbed by soil constituents, the
SMB measurement is often uncertain due to storage conditions, season of collection, low and

irregular recovery, and weak correlation to SMB measured (1).

f) Phopholipid Fatty Acids Extraction Method

This method is based on extraction of phospholipid fatty acids (PFLA) from soil microbial

cell membranes by suitable extractants (11, 14). The lyophilized soil is extracted with the single-

phase CHCl3-CH3OH-buffer system followed by analysis of PFLA by a capillary gas

chromatography with flame ionization detection to SMB (14).

1 nmol PFLA/g soil = 2.4 µg SMB

Extracts are easily analyzed to identify the different PLFA’s for determination of SMB (5)

and but this is a time consuming, complex, and expensive method.

g) Substrate-Induced Respiration (SIR) Method

The SIR method is first reported by Anderson and Domsch (10), which utilizes the initial

change in the soil respiration response as a result of adding glucose or sucrose and nutrients. The

SMB is calculated from the maximum initial respiratory response (MIRR) measured at 22oC as

follows:

SMB = (40.04 x MIRR) + 0.37

Where MIRR is µL CO2/g soil.

This is a fast method to measure SMB but may often overestimate by measuring glucose

responsive active portion of the SMB. The use of glucose may shut down the metabolism of other

microbes. The SIR method requires a GC to measured evolved CO2.

III. UV- SPECTROSCOPIC METHOD

A rapid and inexpensive method to estimate SMB based on the near UV light absorption

of certain molecules (nucleic acids/nucleotides) of microbial cells extracted from CHCl3

fumigated soils (3). The UV absorbance at 280 nm is significantly correlated to SMB measured

by CHCl3 method as follows.

SMB = 21747 x E280 nm

This is a fast and simple method to determine SMB but uses CHCl3. The results are often

compromised from soil colloidal interferences and electrolyte precipitation (3).

CONCLUSIONS

Common and new methods have been discussed but their advantages and disadvantages mentioned.

Over the years, the indirect methods have been more widely used compared to direct methods for

simple, rapid and precise measurement of SMB. Among the indirect methods, the CHCl3

fumigation incubation has been used as a baseline for calibrations and correlations for other

methods. The extraction methods, especially MW soil extraction is gaining increasing acceptance

for its simplicity, rapidity and precision to determine SMB. No method is universally accepted

because of various limitations. There is consequently a demand for further improvement in SMB

measurement.
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