Measuring microbial biomass carbon by direct

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Measuring microbial biomass carbon by direct extraction – Comparison with chloroform fumigation-extraction Article in European Journal of Soil Biology · November 2012 DOI: 10.1016/j.ejsobi.2012.09.005

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European Journal of Soil Biology 53 (2012) 103e106

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Original article

Measuring microbial biomass carbon by direct extraction e Comparison with chloroform fumigation-extraction Raj Setia*, Suman Lata Verma, Petra Marschner School of Agriculture, Food and Wine, The Waite Research Institute, The University of Adelaide, Adelaide SA5005, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 August 2012 Received in revised form 12 September 2012 Accepted 18 September 2012 Available online 3 October 2012 Handling editor: Yakov Kuzyakov

Soil microbial biomass, a small and highly dynamic organic matter pool, plays a critical role in soil fertility. Therefore it is important to have an accurate and rapid method to measure microbial biomass carbon (C). The chloroform fumigation extraction (CFE) method is used by most researchers, but it is quite time-consuming. The direct extraction method where the chloroform exposure and extraction steps are combined is quicker but not often used because it is not clear if it is as reliable as the CFE method. Using 20 Australian soils with a wide range in soil properties, we measured microbial biomass C with the CFE and the direct method. Chloroform labile C extracted by the two methods was correlated (r ¼ 0.87), but it was significantly (p < 0.05) higher with the direct extraction method compared to CFE. Chloroform labile C extracted by both methods was significantly (p < 0.05) positively correlated with clay content, but the correlation coefficient was higher with the direct extraction method. The coefficient of variation for chloroform labile C was greater with the CFE than with the direct extraction method. Chloroform labile C extracted by the direct extraction method did not change between 0.5 and 4 h of shaking with K2SO4 solution and chloroform. We conclude that the chloroform labile C concentrations measured with the CFE method are comparable with those determined by the direct extraction method which is quicker and has a lower variability among replicates. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Chloroform fumigation Clay content Direct extraction Microbial biomass

1. Introduction Soil microbes represent only 5% of the organic matter but play a critical role in soil fertility by synthesis and mineralisation of organic compounds [1]. It is therefore important to determine their activity as well as biomass turnover. Microbial activity can be measured by a range of methods, such as respiration, enzyme activity or gene expression. For microbial biomass on the other hand, only a few methods are used, for example, chloroform fumigation extraction [2], substrate induced respiration [3] and phospholipid fatty acid analysis [4,5]. Among these, the chloroform fumigation extraction (CFE) method is the most commonly used. In this method, soils are exposed to chloroform vapour for 24 h or longer to lyse the microbial cells. Then the fumigated and nonfumigated controls are extracted with 0.5 M K2SO4. The difference between fumigated and non-fumigated carbon (C) is a measure of the chloroform labile C which is then multiplied by a factor to give microbial biomass C. However, chloroform labile C

* Corresponding author. Tel.: þ61 8 8313 7306; fax: þ61 8 8303 6511. E-mail address: [email protected] (R. Setia). 1164-5563/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejsobi.2012.09.005

from non-living sources such as plant residues may also be included and thus lead to an over-estimation of microbial biomass C [6]. Further, this method relies on the complete permeation of the soil sample by the chloroform vapour during the fumigation period. However, diffusion of chloroform vapours into the soil may be affected by texture [7] and organic carbon content [8]. To overcome some of the limitations of the CFE method, Gregorich et al. [8] suggested a direct extraction method for measuring microbial biomass C in which soil extraction and chloroform exposure steps are combined. They found that the efficiency of extraction (as measured by the kEC value) was similar to that of the CFE method of Vance et al. [2]. However, only a few studies have used this method since then [9,10] which may be due to the uncertainty about the reliability of this method. Gregorich et al. [8] used soils with a limited range of pH values (6.5e7.2), organic carbon (14e32 mg g1 soil) and clay contents (19e38%). Further, Gregorich et al. [8] used a shaking time of 30 min, therefore the influence of shaking time on C extraction in presence of chloroform remains to be tested. A further uncertainty about the direct method arises from the fact that chloroform is hydrophobic which may restrict its effectiveness when it is added to a solution during the extraction. Hence, more studies are required so that researchers can make informed decisions about which method is more appropriate.


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R. Setia et al. / European Journal of Soil Biology 53 (2012) 103e106

In this study, we compared chloroform labile C concentrations derived from the CFE and the direct extraction method in 20 Australian soils with a wide range of pH values, texture and organic matter contents. Additionally, we tested the effect of shaking time in the direct extraction method on fumigated and non-fumigated C concentration.

percentages in light-textured and lower percentages in clay-rich soils (Table 1). The required amount of reverse osmosis water was applied gradually to the surface of the soils using a circular motion to ensure homogeneity of wetting. The wetted soils were incubated in the dark for 14 days at 25 C. Throughout incubation, reverse osmosis water was added on a mass basis to maintain the target water content.

2. Materials and methods

2.3. Measurement of biomass C

2.1. Soils

At the end of the incubation, microbial biomass C was measured by the CFE method of Vance et al. [3] and the direct extraction method of Gregorich et al. [8]. For the CFE method, three subsamples of 5 g of each soil were fumigated with ethanol-free chloroform for 24 h at 25 C in a sealed desiccator in the dark. The non-fumigated three subsamples of 5 g of each soil were stored at 8 C during this period. The next day, after removing the beaker of chloroform, the desiccator was evacuated six times to remove the chloroform from the soils. Both fumigated and non-fumigated subsamples were extracted with freshly prepared 0.5 M K2SO4 at a 1:4 ratio and filtered. In the direct extraction method, six subsamples of 5 g of each soil were weighed separately into 50 ml centrifuge tubes and 20 ml of 0.5 M K2SO4 was added to each. To three subsamples, 0.5 ml of ethanol-free chloroform was added. Both the chloroform-exposed and the non-fumigated samples were capped and shaken simultaneously for 1 h. After shaking, the suspensions were allowed to settle for 10 min and the supernatants were filtered through Whatman No. 42 filter. For the sub-samples with chloroform, only the top 15 ml of the supernatant was filtered to reduce the amount chloroform in the filtrate. Filtrates from soils with and without chloroform were immediately bubbled with air for 30 min to remove any residual chloroform. Blanks were treated in the same manner. To assess the impact of shaking time on chloroform labile C determined by the direct extraction method, four soils (3, 10, 19 and 20) covering range of clay and organic carbon contents were treated as described above, but the shaking time was 0.5, 1, 2, 3 and 4 h. Dissolved organic carbon in all filtrates was determined after dichromate digestion by titrating with 0.033 M acidified ferrous ammonium sulphate [14]. Chloroform labile C was calculated as the difference between the C extracted from the chloroform fumigated and the non-fumigated sample. All results are expressed on an oven-dry basis. No conversion factor (kEC) was used to convert chloroform labile C to microbial biomass C because the range of kEC values (0.41e0.58) is used in the literature and it has not been tested which is best suited for the soils used here. Chloroform labile C determined by the two methods was grouped into three groups based on clay content (30%) and the average values for three classes were calculated.

Twenty soils (numbered 1e20) were collected in five Australian states to provide a wide range in pH values, textures and total organic C contents (Table 1). These were sampled to a depth of 10 cm after removing the top 5 cm of a soil. At the time of sampling, most of the sampling sites were cropped except the sites 9 and 11 which were under native shrubs. The soils were air dried and sieved to 2 mm. Air-drying may kill soil microbes, but Australian soils often remain air dry for several months during summer, therefore air-drying is not un-natural. The pH was measured in a 1:5 soil: water suspension after 1 h shaking. Water holding capacity (WHC) was measured using a sintered glass funnel connected to a 100 cm water column (Jm ¼ 10 kPa). The soil was placed in rings in the sintered glass funnel, thoroughly wetted and allowed to drain for 48 h. Dry weight of the soil was determined after oven drying at 105 C for 24 h. Soil texture was determined by mid infra-red spectroscopy on tungsten-milled air dried soils [11] and total organic C content with a LECO C-144 C analyser as described by Merry and Spouncer [12]. 2.2. Incubation To reactivate the soil microbes, the soils were incubated at the percentage water holding capacity optimal for microbial activity as suggested by Setia et al. [13] who determined this value for a range of soils differing in texture and organic carbon content. The optimal value varied from 45 to 80% of water holding capacity, with higher Table 1 Properties of the soils and percentage of water holding capacity during incubation prior to extraction. Soil

Site

pH

Clay %

Organic carbon mg g1 soil

Maximum WHC g g1 soil

WHC during incubation %

1 2 3 4 5 6 7 8 9 10 11

Riverway, NSW Lochslea, NSW Kelley, NSW Tudgey, NSW Culcairn, NSW Mount Tyson, QLD Stansbury, SA Hart, SA Kadina, SA Monarto, SA Lake Alexandrina, SA Warramboo, SA Monarto, SA Hopetoun, VIC Birchip, VIC Walpeup, VIC Horsham, VIC Wongan Hills, WA Fleming, WA Boathaugh, WA

8.3 8.0 8.2 8.0 5.9 7.7 5.8 7.8 7.8 8.2 3.8

28 21 53 49 11 56 12 32 40 8 1

12.2 10.2 11.8 16.4 12.5 20.1 7.8 15.9 20.6 8.2 1.8

0.43 0.17 0.66 0.59 0.27 0.37 0.13 0.33 0.43 0.16 0.08

50 55 40 40 80 40 55 50 40 80 80

8.5 8.4 8.5 8.1 8.1 8.3 8.2 6.0 6.1

13 18 14 45 9 40 48 3 22

9.9 19.6 8.5 10.2 6.3 10.7 18.7 6.3 14.6

0.20 0.29 0.16 0.30 0.12 0.42 0.33 0.08 0.26

80 55 55 40 80 40 40 80 50

12 13 14 15 16 17 18 19 20

NSW ¼ New South Wales, QLD ¼ Queensland, Vic ¼ Victoria, WA ¼ Western Australia, SA ¼ South Australia. pH was measured in 1:5 soil: water ratio, WHC ¼ water holding capacity.

2.4. Statistical analyses A t-test was used to determine if there was a significant difference in non-fumigated and fumigated C extracted between the two methods. The relationship between non-fumigated C, fumigated C and chloroform labile C by the two methods and soil properties was analysed by correlation coefficient (r) and stepwise regression using SPSS-VÒ (10.0) software. Significant differences between chloroform labile C at different clay contents and shaking times were analysed by two way ANOVA with unbalanced design and one way ANOVA, respectively. Means were compared by using Duncan’s Multiple Range Test with P 0.05 (GenStat for Windows 11.0, VSN Int. Ltd, UK, 2005). Principal components analysis (PCA) was used to plot differences among the soils with respect to pH, clay, organic carbon and chloroform labile C extracted by two extraction methods (Primer-E Ltd, Plymouth Marine Laboratory, Plymouth, UK).



3. Results

105

30

Direct CFE

25

20

CV(%)

In the 20 soils, the pH varied from 3.8 to 8.5, the clay content from 0.5 to 56% and the organic C content from 1.8 to 20.6 mg g1 soil (Table 1). The organic C content was significantly (p < 0.05) positively correlated with clay content (r ¼ 0.68) but not with pH. The C extracted from the fumigated soils ranged between 89 and 385 mg kg1 soil by the CFE method and between 138 and 483 mg kg1 soil by the direct extraction method (Table 2). The C extracted from the non-fumigated soils varied between 10 and 119 mg kg1 soil by the CFE method and between 10 and 155 mg kg1 soil by the direct extraction method. Both fumigated and non-fumigated C measured by the two methods were significantly (p < 0.05) positively correlated (r ¼ 0.96 and 0.90 for nonfumigated C and fumigated C, respectively). The chloroform labile C varied between 78 and 330 mg kg1 soil by the CFE method and between 128 and 401 mg kg1 soil by the direct extraction method and it was correlated (r ¼ 0.86) between the two methods. Chloroform labile C was significantly higher with the direct method compared to the CFE method (Table 2) and the coefficient of variation in chloroform labile C for the majority of soils was lower with the direct extraction method (0.7e16%) than with the CFE method (1.4e25%) (Fig. 1). The relationship between fumigated, non-fumigated and chloroform labile C measured by both methods and soil properties was assessed by stepwise regression. Among three soil properties measured, only clay content was significantly correlated and explained 49, 26 and 32% of the variance in fumigated, nonfumigated and chloroform labile C extracted by the direct method, respectively. For the CFE method, it explained 37, 24 and 21% of the variance, respectively. Chloroform labile C extracted by the two methods was significantly higher in soils with >30% clay compared to soils with lower clay contents and the variability in chloroform labile C concentration at all clay contents was higher with the CFE method (Fig. 2). The chloroform labile C concentration (mg kg1 soil) did not vary significantly with shaking time: 0.5 h 295, 1 h 322, 2 h 326, 3 h 328 and 4 h 305. Similarly the non-fumigated C concentration (mg kg1 soil) did not change significantly during shaking : 0.5 h 28, 1 h 28, 2 h 31, 3 h 31 and 4 h 32.

15

10

5

0 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

Soils Fig. 1. Coefficient of variation (CV) of chloroform labile C measured with the chloroform fumigation extraction (CFE) and the direct extraction method in the 20 soils (n ¼ 3).

The PCA based on the measured soil properties and chloroform labile C extracted by both methods showed that 99.8% of the variation could be explained using two principal components (Fig. 3). The first PC (PC1) accounted for 95.8% of the total variability and was associated with labile C (eigenvector ¼ 0.998) while the second PC (PC2), representing 4% of the total variance, was mainly correlated with clay content (eigenvector ¼ 0.975). The chloroform labile C concentration was 300 mg C kg1 soil for the soils in the middle. The difference in chloroform labile C extracted by both methods was greater for soils 4, 6, 8, 13 and 17 (Table 1 and Fig. 3) than in the other soils. 4. Discussion This study showed that the direct method yields similar concentrations of chloroform labile C as the CFE method and is quicker and less variable than the latter. Fumigation extraction is

Table 2 Fumigated, non-fumigated and chloroform labile C extracted with the direct method or the chloroform fumigation extraction (CFE) method. Values are means (n ¼ 3) SE. Soil

Direct method Fumigated C

CFE method Non-fumigated C

Chloroform labile C

Fumigated C

Non-fumigated C

Chloroform labile C

mg kg1 soil 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

419 191 430 456 258 483 219 402 376 349 317 400 386 241 319 174 355 410 138 301

4.8 3.2 5.6 6.7 5.8 1.6 5.4 6.1 2.8 3.4 1.2 3.2 10 6 14 4.6 19 0.01 1.2 11

44 54 50 113 34 83 52 90 113 20 80 34 21 68 27 36 25 155 10 47

CFE ¼ Chloroform fumigation extraction.

3.6 6.6 2.6 2.5 2.5 0.01 11.3 9.5 4.2 6.2 7.1 0.01 2.0 4.9 7.4 3.8 6.0 7.5 2.2 3.5

375 136 380 343 223 401 167 312 263 329 237 366 365 173 293 138 330 255 128 253

1.6 6.3 4.2 7.7 3.4 1.6 15.7 9.9 41 9.7 6.7 3.2 8.7 10 8.4 8.3 7.5 7.5 1.6 14

367 184 385 365 237 291 159 339 354 316 300 274 303 214 260 157 274 350 89 256

19 3.2 13 8.8 1.8 2.2 5.7 2.3 8.1 5.5 7.0 5.8 4.6 6.9 11 13 84 2.2 2.0 6.2

42 56 55 95 18 89 52 84 116 19 84 34 17 52 23 38 10 119 12 46

3.3 8.2 2.6 1.9 3.5 7.8 9.8 8.9 7.4 3.5 1.5 2.6 1.8 4.4 4.6 4.1 2.2 7.6 0.6 1.5

325 128 330 270 220 201 107 255 237 297 216 239 286 162 238 119 264 232 78 211

16 6.0 13 11 1.8 6.6 15 8.5 9.8 8.2 6.0 8.2 2.9 2.6 10 13 9.5 9.8 1.6 5.2


106


Direct CFE

500

-1

Chloroform labile C (mg kg soil)

600

400

300

200

100

0 30

Clay (%) Fig. 2. Microbial biomass C measured by chloroform fumigation extraction (CFE) and direct extraction methods in three clay content classes. Vertical lines indicate standard error of the mean.

penetrating the soil and coming in direct contact with the microbes during shaking. Clearly, strong shaking is essential for this method. Compared to CFE method, there was a stronger relationship in the direct extraction method between chloroform labile C and clay content (Fig. 2). This can be explained by the fact that in clay-rich soils with their large proportion of small pores which are often water-filled, may limit the permeation of the soil by the chloroform vapour in the CFE method. This is also supported by the PCA which showed that more chloroform labile C was extracted with the direct method in soils 4, 6, 8, 13 and 17 and these soils are characterised by high clay and high organic carbon content. Further advantages of the direct method include (i) the lower variability of chloroform labile C among replicates (Fig. 1) and (ii) less time required. Our results show that with the direct method, a shaking time of 30 min was sufficient since there was no significant change in chloroform labile C concentration from 0.5 to 4 h of shaking. We conclude that the direct extraction of non-fumigated and chloroformfumigation with 0.5 M K2SO4 solution for 30 min is a rapid alternative method for quantifying microbial biomass C in soils. Acknowledgements

Method 200

Direct CFE

We thank Dr Sean Mason, Dr Sam Stacey and Mrs Deepika Setia for providing the soils used in this study.

PC2

References

19 0

19 7 16 7 14 16 14

2 2

8 pH OC

10 13 12 Labile C 4 17 3 6 3 11

12 11 11 5 10 13 8 5 15 4 15 9 20 17 9 18 6 18 20

Clay

-200 -400

-200

0

200

400

PC1

Fig. 3. Principal component analysis on pH, clay, organic carbon and chloroform labile C measured with the chloroform fumigation extraction (CFE) and the direct extraction method.

the most wide-spread method for determination of microbial biomass C, but it has a number of limitations which can result in erroneous values, particularly for inexperienced users. The method relies on consistent creation of chloroform vapour which may not be always the case, for example, if the desiccators are not completely sealed or the applied vacuum is insufficient. And as outlined in the introduction, high clay or organic matter content may also limit the permeation of the soil by the chloroform. These limitations are largely overcome in the direct method because the soils are shaken with a solution containing chloroform. However, one reason why researchers have not considered this alternative may be the concern that the effectiveness of the chloroform is reduced because it is hydrophobic and therefore does not dissolve in the 0.5 M K2SO4 solution, instead it forms an emulsion. Our results indicate that this is not the case since the chloroform labile C concentrations were higher with the direct than the CFE method. This can be explained by droplets of chloroform

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