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Estimation of Soil Microbial biomass using Substrate Induced Respiration: An Experimental Review Study with Loamy Soil of North... Conference Paper · June 2014 DOI: 10.13140/2.1.2139.9049

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Estimation of Soil Microbial biomass using Substrate Induced Respiration: An Experimental Review Study with Loamy Soil of North Bangalore* Anshuman Swaina, Abhishek Bastiraya, Kethepalli Jitendraa, Ramakrushna Haibrub

a b

Indian Institute of Science (IISc), Bangalore, India-560012;

National Institute of Science Education and Research (NISER), Bhubaneswar, India-751005

Abstract A standardization protocol was designed for measurement of the microbial biomass in the soil using the substrate induced respiration (SIR) method. Each part of the standard protocol usually followed in the SIR (Anderson and Domsch, 1978) was reviewed and certain changes were made keeping in mind efficacy in result & analysis and also easier laboratory execution. Throughout the study D-glucose was used as the primary substrate. Fresh soil samples were used for the study (after determining certain physical properties) which involved determination of respiration processes across varied glucose concentrations for fixed time, and that of across varied time durations for same substrate concentration, in order to test the validity of the process designed. Key Words: Substrate Induced Respiration, Microbial Biomass

as a measure of computing the microbial

Introduction

activities in soil. This process was designed

The method of substrate induced respiration

quick assessment of live microbe carbon

(SIR) is used to estimate the extent of soil respiration due to the addition of a substrate such as glucose, glutamic acid, mannitol and amino

acids.

This

technique

uses

the

physiological respiration reactions of the organisms that are present in the soil towards

substrate

addition,

such

as

production of CO2 and/or consumption of O2 --------------------------------------------------------*Done under the supervision of: Dr Sumanta Bagchi, Asst. Professor, CES, IISc, Bangalore, India-560012

by Anderson and Domsch in 1978 to offer a biomass in soils. The substrates that can be employed are not restricted to the aforesaid ones only; the choice of the substrate that is used should reflect the organisms that are in target and soil type that is being tested. Though other substrates can be used to produce measurable respiration glucose was found

to

produce

the

maximal

results

(Anderson and Domsch, 1978). The method of Substrate induced respiration has also been utilized in combination with solutions intended to behave as selective inhibitors, 1

such

as

cycloheximide

(fungicide)

or

streptomycin sulphate (bactericide), to limit the certain specific microbial populations (Lin and Brookes, 1999). This technique can be finished quickly and has a low analysis time, depending on the apparatus used to measure respiration. The results that were achieved by the revised SIR method also seem

to

compare

well

with

other

conventional biomass estimation procedures such as chloroform fumigation incubation (CFI)

(Jenkinson

and

Powlson,

1976),

chloroform fumigation extraction (Vance et al. 1987) etc…

2. Determination of certain physi-cal properties of the soil Certain physical properties of the soil were determined such as pH, water holding capacity and the soil texture. The soil texture was determined just to know the type of the soil, but the other two are quite important for the primary experiment itself. If the pH is more than 6.5, then the soil will be able to hold CO2 in itself, and thus the data obtained has to be revised upon using some other techniques, and if it is less than pH 6, then no such problem arises. The water holding capacity needs to be known because the substrate can be more easily distributed evenly in the soil if it is given in

Method 1. Collection and processing of the Soil sample The soil that is meant to be tested is collected and transported in such a way that its properties remain intact. It is not stored

in

an

ambient,

air-circulating

environment, if it is intended for immediate use, as in the case of this study. It is usually prepared by sieving the soil in order to remove living plants, nematodes, annelids and other undesirable organic constituents. These materials can influence vital parameters, like CO2 absorption and O2 production, and thus affecting accurate measurement of microbial respiration; thus they must be removed. Sieving the soil, later, also allows an even spreading of added substrate which in turn produces a more dispersed release of the produced CO2 (Sparling, 2005).

the solution phase. Moreover if it is given in the solution phase, the amount of solution added should not exceed the water holding capacity of the soil, otherwise it will create other problems like anaerobic conditions, unavailability of all glucose, change in the main media property itself etc…

3. Addition of the substrate and the main setup The soil samples were measured at 10gm and put in a conical flask, then glucose of a certain concentration was added to it at 50% water holding capacity, and the mouth of the flask was corked and there was a tube

through

the

cork

connecting

to

another similar flask tilted horizontally at a higher elevation that containing 10 mL NaOH solution of a certain concentration. The CO2 that will be released by the microbes in the flask will be absorbed by the NaOH solution in the other flask.

2

4. Determination of the amount of CO2 released

water holding capacity of 3.5 mL/10 g of

The amount of CO2 released and that has been absorbed by the NaOH solution, is determined by titration against HCl of

soil. It was freshly collected and used, thus did not require any special incubation or other treatment.

Figure 2 Complete Experimental Setup

The first part was measurement of CO2 released by varying glucose concentration from 10mg/1g of soil to 70mg/1g of soil, keeping

Figure 1 Setup of the flasks

the

total

time

of

incubation

constant at 5 hours. Then after obtaining

known concentration after addition BaCl2 or Ba(NO3)2 in order to precipitate the

the graph, the glucose concentration value where the CO2 released was maximal, was

carbonate, and using phenolphthalein as the

chosen, and keeping it constant the amount

indicator.

the

of CO2 released was found out by varying

amount of original NaOH that was left.

the time from 0 hour till 10 hours in

Hence subtracting the value from the

intervals of one hour each.

original amount given, will give us the

Each sub-part was repeated at least six

amount of carbonate formed i.e. the amount

times in order to get the final statistical

of CO2 absorbed after comparing with a test

result.

The

reading

will

show

solution which will be an NaOH solution freshly

prepared.

As

we

consider

the

amount of CO2 released as the measure of microbial biomass, we can estimate the microbial biomass in terms of it.

Results The results of the first part of the experiment involving the determination of the concentration at which there is maximal consumption, is as follows, represented in

Experiment The above protocol is followed throughout the experiment involving the loamy soil collected from a site in Northern Bangalore

Table 1 and Figure 3. All the mentioned data are the statistical average of at least 6 readings of the same experimental setup.

(Karnataka, India). It had pH 8.34 and a 3

0

Average Volume of HCl used per 10mL NaOH (in mL)

0 0.25 0.38

9.25

0.1

9.00

0.3

8.6

0.2

8.87

0.4

8.57

0.5

8.95

0.7

8.93

0.6

Difference from the test solution (in mL)

0.65 0.68 0.3 0.3 0.32

8.97

CO2 released above the basal level (in proportional units)

Glucose Concentration (in g/10g of soil sample)

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.2

0.4

0.6

0.8

Glucose Concentration (in g/ 10 g of soil sample) Figure 3

Table 1

After obtaining the maximal amount of

at different time intervals, all of whose data

glucose consumed, the same concentration

is presented in Figure 4 and Table 2.

is used for observing the microbial biomass

Time (in hours)

Volume of HCl used per 10 mL of NaOH (in mL)

Difference from the test solution (in mL)

1

9.5

0.6

3

9.3

0.8

5

8.9

7

8.6

9

8.2

0

10.1

2

9.4

4

9.2

6

8.6

8

8.3

10

8.1

0

0.7 0.9 1.2 1.5 1.5 1.8 1.9 2

Table 2 4

The difference from the test solution here

concentration of glucose that was available

corresponds directly to the amount of

to the microbes for respiration was lower

carbon dioxide released an absorbed in the

than

form of carbonate.

increased, the glucose available per capita,

it.

Thus,

as

the

concentration

2.5

CO2 released above the basal level (in proportional units)

2

1.5

1

0.5

0 0

2

4

6

8

10

12

Time in hours Figure 4

So, this gives us a direct proportional unit of the amount of carbon dioxide released.

also

increased

that

led

to

increased

respiration and hence, more CO2 released. But after the mentioned concentration is a decline and stabilisation of the CO2

Discussion

released. This decrease is due to decreased

The microbes present in the soil consume

biomass activity. This can be explained by

Glucose and produce both CO2 as a

the fact that after 0.4 g/10g of soil, the

product of respiration.

glucose concentration starts becoming very

C6H12O6+12O2 6CO2 + 6H2O + Energy

high. In such a case, Crabtree effect comes

The amount of CO2 released is a direct

the glucose anaerobically and hence the

measure of the amount of microbial biomass in the soil. So, in the first part of the

experiment,

we

found

that

the

maximal respiration was found to be at 0.4g/10g of soil. Below the mentioned concentration, the

into play and the organisms start using CO2 emission decreases. Moreover the fall is

gradual

because

the

concentration

beyond which the Crabtree Effect happens is different for various organisms living in the soil. The stabilisation can be explained using

the

point

that

the

glucose

amount of respiration was lower as the 5

consumption reaches a saturation level

there for the rest of the microbes that still

don’t undergo the effect. This is seen in

than

Figure 3. For the second part of the

absorption of the CO2 by the soil can be

experiment, a glucose concentration of

ignored without affecting the result by a

0.4g/10g of soil was chosen and the values

statistically significant margin.

of CO2 released after every hour was seen.

analysis done previously still holds.

The plotted graph from the data (figure 4)

The quantitative amount of microbes now

actually matches the synchronous culture growth curve of multiple generations, as the total number of microbes increase in the expected exponential way.

moderately alkaline in nature. But the qualitative results that were shown above don’t change. In alkaline soils, a certain amount of CO2 is absorbed by the soil and as it is fixed for a given time, it is taken care of by the baseline used (test solution) in the first part. In the second part even though the CO2 absorbed changes with time, it is absorbed at a constant pace and thus do not alter the nature of the graph

in

a

great

way.

Moreover the amount of CO2 absorbed is quite

less

compared

to

µmol/hour)

the

amount

released by the microbes. But in order to determine the quantitative amount of microbial biomass, the absorbed amount of CO2 must also be considered.

and

thus,

the

So, the

can be calculated from the second curve (figure 4). Assuming there is binary fission only for most cases and the second generation

But a problem arises as the soil is

exponential

4

median

respiration

rate

is

measured at 3 hours & third generation median is at 6 hours and it is proportional to the total microbial biomass at that time, the initial biomass can be calculated by the following: Initial biomass emission=z Median Biomass emission at first generation=A Median Biomass emission at second generation=B So, knowing that the number of organisms is just double at second generation median as compared to first generation, we get: 2(𝐴 + 𝑧) = 𝐵 + 𝑧 ⇒ 𝑧 = 𝐵 − 2𝐴

Thus the value of z, in proportional units, after substituting the values of A=0.7 and B=1.5, we get z=0.1 proportional units. Now, the proportional units signify the

About 0.1–0.5 µmol m-2 s-1 was noted for all alkaline soils all over the world** (Xie et al, 2009) and using this data, we can establish a limit for the amount of biomass

volume (in mL) of 0.04N HCl used for titration. Hence the value of biomass is 4 µmol C/10g soil, which is equal to 4.8 µg C/1g of soil.

present in the soil. So, for the 10g of soil sample that was taken in each of the conical

flasks,

the

amount

of

**This is calculated for a soil depth of 15 cm.

CO2

absorbed comes in the range of 2-50 Picomoles per second or 7-180 nano-moles per hour. And the CO2 released per hours is in the range of micromoles (at least greater 6

measurement of microbial biomass in

Conclusion The standardised protocol of soil biomass determination using the substrate induced respiration displays all the results that are known to be true from other confirmed experiments and hence

its value

for

biomass determination would be correct. Moreover it also allows us to see multiple generation growth in soil microbes and also a demonstration of the Crabtree effect. In short, SIR can be a good and easy measure of the soil microbial biomass and other aspects of microbial growth in the soil.

Journal of Microbiological Methods 5, 1986, pp. 177 – 189 4. Anderson

JPE,

and

Domsch

H

(1973), “Quantification of bacterial and

fungal

contributions

to

soil

respiration”, Arch. Mikrobiol. 93, pp. 113-127 5. Anderson JPE and Domsch

KH

(1978), “A physiological method for the

quantitative

measurement

of

microbial biomass in soil”, Soil Biol. Biochem. 10, pp.215- 221 6. Cook FJ and Orchard VA (1984), “Relationship

Acknowledgements We are grateful to Dr Sumanta Bagchi for giving us an opportunity to work in his lab and also to Mr Manjunatha H C, for his great help in setting up the experiment and to other people in his lab for their sincere cooperation.

1. Ananyeva

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soils

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99- 104 ND,

Susyan

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Respiration”,

Eurasian Soil Science, 2011, Vol. 44, No. 11, pp. 1215–1221. 2. Blagodatsky SA, Heinemeyer O, and Richter J (2000), “Estimating the and

biomass

between

glucose”, Soil Biol. Biochem. 13, pp.

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