Full Length Research Paper
Microbial biomass carbon determination by chloroform
fumigation and substrate induced respiration methods
S. Asuming-Brempong and E. O. Odei
Department of Soil Science, University of Ghana, Legon *Corresponding Author’s Email: sbrempong@yahoo.com
Abstract
Microbial biomass is a nutrient pool, driving force of nutrient turnover and early indicator of soil/crop management that responds very quickly to changes in crop management practices. The objective of the study was to determine the microbial biomass of soil by the chloroform fumigation method (CFM) and the substrate induced respiration (SIR) and also to assess the soil quality. Microbial biomass carbon by CFM ranged from 161.9 to 296.0 ug CO2-C/g soil whilst biomass determined by SIR ranged from 195 to 300 ug CO2-C/g soil. Generally, microbial biomass decreased with soil depth. The Cmic/Corg (microbial quotient) values ranged from 4.5 to 9.00 % in the Adenta series whilst that of the Haatso series ranged from 9.6 to 16.60 %. A significant correlation of 0.86 was obtained between the basal respiration and available soil P. The basal respiration and the organic carbon content of the Typic Kandiustalf (Adenta series) were higher than that of the Haatso series (Quazisament), suggesting that the Adenta series has a higher fertility status than Haatso series.
Keyword. index: microbial biomass, chloroform fumigation, substrate induced respiration.
INTRODUCTION
Microbial biomass is the part of organic matter in soil that constitutes living microorganisms smaller than 10 µm3. Fungi, bacteria and archae, protists, meso and macro-fauna (microarthropods, macroarthropods, enchytraeids) constitute the total living biomass in soil, whilst the earthworm form only a minor fraction (Beare, 1997). Typically, microbial biomass carbon ranges from 1 to 5% of soil organic matter (Sparling, 1985; Smith and Paul, 1990).
Microbial biomass carbon has been found to be related to aggregate stability of soil and the effect of toxic materials such as pesticides and heavy metals in soil (Haynes and Swift, 1990; Anderson et al. 1981). Some enzymes in the soil such as dehydrogenase, invertase are also related to microbial biomass carbon (Burns, 1978). Management practices such as long – term fertilization and organic manure use had a significantly great impact on the microbial biomass carbon and dehydrogenase activity (Chu et al, 2007). But, Nannipierri
et al (2002) showed that enzyme assays were often poorly correlated with microbial biomass size, even when
reasonably correlated with activity and hence were not likely to give reliable estimates of the size of microbial biomass. The size of microbial biomass was strongly correlated with content of base cations, base saturation, cation exchange capacity and organic matter quality of soil (Zwolinski, 2002). Results of Wolinska et al (2012) showed significant positive relationship existing between DNA and soil microbial biomass content but negative correlation existed between microbial biomass, water potential (pF) and oxygen content (ODR) of soil. These findings point out a distinct relationship between soil fertility and soil microbial biomass suggesting that microbial biomass measurement provide a valid estimate of soil quality. Forest types affected soil microbial properties significantly due to the differences in soil physical –chemical features (Cheng et al. 2013).
Any existing relationship between microbial biomass carbon and biodiversity in the soil has not been clearly established hence one cannot conclude that soils with high microbial biomass carbon have a high biodiversity. Also, no consistent trends between reducing biodiversity
and alterations in functions have been observed (Griffiths
et al. 2001). Other studies have shown that reducing biodiversity increases the variability of functional
measurement within systems (McGrady-Steed et al,
1997; Naeem and Li, 1997).
The proportion of microbial biomass carbon in total soil organic carbon is the microbial quotient that gives insight into the capability of a soil to support microbial growth and it is expected that soils with high quality maintained higher ratios of the microbial quotient (Insam and Domsch, 1988). The metabolic quotient (qCO2) is the
ratio of basal respiration (BR) to the total microbial biomass carbon (Insam and Haselwandter, 1989). The
qCO2 indicates the efficiency by which soil
microorganisms use C-resources in the soil and it is expected that stressed soils will provide higher qCO2
values than less-stressed soils (Insam and Haselwandter, 1989).
Microbial biomass is a nutrient pool that responds quickly to changes in crop management practices or environmental conditions than soil organic matter (Dick, 1992, Shibahara and Inubushi, 1997). As a useful monitoring index, the microbial biomass carbon was used to monitor soil quality, when straw was incorporated into the soil (Powlson et al. 1987).
Many methods have been developed to measure microbial biomass and one of such rapid methods was by Anderson and Domsch (1978) the substrate induced respiration (SIR) method that allowed the estimation of the amount of carbon held in non-resting, living microorganisms in soil sample. The initial respiratory response to added glucose, recorded before any development in existing soil microflora could be viewed as an index of the existing soil microflora (Anderson and Domsch, 1978).
The relationship between the SIR method and other biochemical estimates of microbial biomass has been reasonably consistent, with anomalies generally being attributed to inaccurate measurement of CO2 and the use
of inappropriate techniques for calibration (Sparling, 1981)
The present study was conducted to determine the microbial biomass of two tropical soils using two different methods of determination and assessing the state of the microbial community.
MATERIALS AND METHOD
Sampling, collection and preparation of soil samples
Adenta and Haatso series (Typic Kandiustalf and Quazisament), were sampled from the University of Ghana farm at three different depths viz (0-20 cm), (20-40 cm) and ((20-40-60 cm). On the Adenta series for instance, an area was demarcated and with an auger
soils were sampled at various places for a particular depth (eg. 0-20 cm). The soil was composited together for that particular depth and labeled and stored in an ice chest. The auger was carefully cleaned with 70 % ethanol and soil of the next depth say 20-40 cm was sampled from various places on that demarcated piece of land. After sampling on that demarcated land on the Adenta series, the soils were composited and mixed well and labeled and stored on ice. In a similar manner sampling was done on the Haatso series for the various depths. The samples were put in an ice chest and immediately brought to the laboratory. The soils were sieved through a 2 mm sieve.
The Adenta and Haatso series form part of the Toje-Adenta-Haatso soil series along a catena down the Legon hill, University of Ghana. The soils were chosen because they occurred extensively on the Accra Plains. The soils were sampled within the coastal savanna zone of Ghana, which is part of the dominant ecosystem of West Africa. The total annual rainfall is about 900 mm and is bimodally distributed with mean monthly maxima in May and October with a relative humidity ranges between 59 % in the afternoon and 93 % at night. The mean annual temperature is 28° C (Dowuona et al. 2012). The vegetation is grassland with substantial amounts of thickets and clumps. The soil samples were sieved using 2 mm sieve and part of the soil was used for the determination of microbial biomass carbon. The rest of the sieved soil was used for chemical analyses.
Chemical analysis of soil samples pH (H2O)
Twenty g of the sieved soil was weighed into a beaker and 40 mL of distilled water added to give 1:2 soil water ratio. The mixture was stirred continuously for about 30 minutes and then left to stand for about 1hr to allow most of the suspended clay particles to settle and also allowed to obtain the room temperature. The glass electrode of the pH meter was standardized using two aqueous solutions of pH 4 and 7 and then later used to measure the pH of the prepared soil sample by carefully immersing the glass electrode into the supernatant.
pH (1 M KCl)
Soil of 10 g was weighed into a beaker and 20 mL of 1M KCl added to give a ratio of 1: 2 soil to KCL solution. The mixture was stirred for about 30 minutes and then left to stand for 1hr to allow most of the suspended clay particles to settle and also allowed to obtain the room temperature. The pH of the prepared soil sample was obtained by carefully immersing the glass electrode into the supernatant.
Available Phosphorus (Bray 1 Method)
Available phosphorus was determined using the method of Bray and Kurtz (1945). Five grams of soil was weighed into a centrifuge tube and 50 mL of 0.03M NH4F in 0.025
M HCl was added. The suspension was shaken for 3 minutes on a mechanical shake. After that, it was filtered. Five mL aliquot of the filtrate was taken into a 50 mL volumetric flask. A yellow colour was developed when a drop of para-nitrophenol indicator was added together with a drop of ammonia solution into the filtrate. Seven millilitres of reagent B, prepared by dissolving 1.056 g of ascorbic acid in 200 mL of reagent A was added to the aliquot and topped up to the 50 mL mark with distilled water. After the colour had developed, its intensity was measured with a spectrophotometer. Reagent A was made by dissolving 12 g of ammonium molybdate in 250 mL of distilled water and adding 0.2998 g of antimony potassium tartarate. The dissolved reagent were added to 1000 mL of 2.5M H2SO4 (148mL conc. H2SO4) and
mixed thoroughly and made up to 2000 mL.
The spectrophotometer was calibrated using standard phosphorus solution. The intensity of the blue colour was measured using the Philip PU 8620 spectrophotometer at a wavelength of 712 nm. A blank solution was prepared with 7mL of reagent B and distilled water and colour was also developed as above. The P content in the sample was calculated.
Total Nitrogen (Kjeldahl Method)
The Kjeldahl method was used for the determination of total nitrogen. The nitrogen in the sample was converted to ammonia by digestion with concentrated H2SO4 using
a nitrogen catalyst (Selenium powder). The ammonium formed was determined by distillation of the digest with an alkali and titrated with a standard acid.
Air-dried soil of two grammes was weighed in triplicate into 300 mL Kjeldahl flask and a few mL of distilled water added to moisten the soil. The nitrogen catalyst was added followed by 20 ml of concentrated H2SO4. The
mixture was digested for at least 30 minutes till it became clear. The digest was allowed to cool, transferred with distilled water into a 100 mL volumetric flask, and made up to the volume. Five millilitres aliquot was pipetted into a Markham distillation apparatus and 5 ml of 40 % NaOH added and rinsed to about 100 ml. Five millilitres of 20 % Boric acid and few drops of a mixed indicator (0.13 g of methyl red plus 0.666 g of methylene blue dissolved in 100ml of 95% ethanol) were put into a conical flask. The distillation process was set up to titrate the trapped NH3
gas which appeared light green at endpoint. The solution was back titrated with 0.01M HCL green to purple endpoint. From the results, the percentage nitrogen in the soil was calculated.
Organic Carbon
Organic carbon was determined using the wet oxidation method of Walkley- Black (1934). This method involved the reduction of Cr ion by the organic matter and the unreduced Cr2O72- measured by titration with Ammonium
Ferrous Sulphate. The quantity of organic matter oxidised is calculated from the amount of Cr2O7
reduced.
One half of a gram of finely ground soils sieved through a 2 mm was weighed in triplicate into 500 ml Erlenmeyer flasks. Ten millilitres of 1.0 M potassium dichromate (K2Cr2O7) followed by 20 ml of concentrated H2SO4 were
added to the soil. The flask was swirled making sure the solution was in contact with all particles of the soil and allowed to stand on asbestos sheets for about 30 minutes.
Then, 200 ml of distilled water was added and this was titrated against 0.5 M acidified Ammonium Ferrous Sulphates. In the titration, 5 ml of 85% orthophosphoric (H₃PO₄) acid and 2 ml of Barium diphenyl-4 sulphonate indicator were added before titrating against the Ammonium Ferrous Sulphate from an orange colour to a green end point. The percent organic carbon was calculated.
Determination of microbial parameters
Microbial biomass carbon was determined for soil samples according to the method of Jenkinson and Powlson (1976). The soil samples for the various treatments were preincubated for 7 days at room temperature at 40 % moisture holding capacity of soil. After that period, chloroform fumigation was carried out. The CO2-C evolved at room temperature (28ᵒ C) over the
0-10 days from both control and chloroform fumigated soils was measured by method described by Anderson (1982). The CO2-C flush data were converted to µg
biomass carbon using a mineralization coefficient (kc) of 0.40 (Jenkinson et al., 1979).
The carbon dioxide evolved from the unfumigated soil after incubating for 10 days and trapped in 1 M NaOH during the chloroform fumigation studies indicated the basal respiration.
The method of Anderson and Domsch (1978) was followed with some modification to determine the substrate induced respiration. The modification involved the use of 10 ml 1M NaOH to trap the CO2 that was
evolved instead of using a gas chromatogram which was not available in the laboratory. The NaOH was titrated against 0.5M HCl after 1.0 ml of 1.5 M BaCl2 had been
added to precipitate the carbonate as barium carbonate.
The substrate induced respiration values were
transformed to biomass carbon using the formula as described by West and Sparling (1986) as follows;
Table 1. Some chemical properties of the Haatso and the Adenta series
Depth (cm) pH (H2O) pH(KCl) Soil organic
carbon g/kg Total nitrogen g/kg soil % Available P Adenta series 0-20 6.00 4.31 5.68 0.85 5.75 20-40 5.50 4.00 4.67 0.56 4.32 40-60 5.80 3.94 2.75 0.74 3.40 Haatso series 0-20 6.01 4.84 2.57 0.45 5.24 20-40 5.40 4.40 1.67 0.34 4.04 40-60 5.40 4.36 1.25 0.21 3.18
Table 2. Microbial biomass determination by chloroform fumigation and substrate induced respiration in the Adenta and the Haatso
series Soil (cm) CFM ug CO2-C/g soil SIR ug CO2-C/g soil Basal respiration ug/CO2-C/g soil qCO2 Cmic/Corg (%) Adenta series 0-20 280 195 72 0.28 4.50 20-40 296 267 52 0.17 6.30 40-60 248 300 44 0.19 9.00 Haatso series 0-20 295 192 64 0.20 11.40 20-40 161 269 52 0.33 9.60 40-60 209 274 35 0.18 16.60 LSD (0.01) 19.13 34.67 15 0.26 6.3
The metabolic quotient (qCO2) was determined as
qCO2 = basal respiration
microbial biomass carbon
The microbial quotient (Cmic/Corg)
= microbial biomass carbon
Soil organic carbon
Data from the various treatments were analyzed by using Genstats (9th edition) statistical software. Analysis of variance (ANOVA) was run on the parameters to ascertain significant differences among treatments. The means were separated using the least significant difference.
RESULTS AND DISCUSSION
The organic carbon content of both soil series was generally low. The Adenta series had higher organic
carbon content (5.68 g/kg soil) almost twice as much as that of the Haatso series for the 0-20 cm depth (2.57 g/kg soil, Table 1). The organic carbon decreased with depth in both soil series. The total nitrogen followed a similar trend being almost twice higher in the Adenta (0.85 g/kg soil) than the Haatso series (0.45 g/kg soil, Table 1) and it also decreased with depth. The soil pH for both soil series was strongly acidic from the surface to very strongly acidic as one moved down the soil profile. The change in pH between that of 1 M KCl and water was negative suggesting that the soil colloidal surface was negatively charged. The available phosphorus was almost the same for both soil series (5.75 % for Adenta series and 5.24 % for Haatso series for the depth 0-20 cm and for the other depths).
The microbial biomass carbon as measured by chloroform fumigation was higher in Adenta than in the Haatso series (Table 2). This could be due to the higher organic matter content in the Adenta series than the Haatso series resulting in higher energy supply for microorganisms especially the heterotrophs in the Adenta series. Microbial biomass carbon decreased with soil depth. In the Adenta series however, the microbial
Table 3. Matrix of correlation of microbial biomass carbon and some other microbial parameters in the Adenta and Haatso series Soil organic carbon Microbial biomass (CFM) Microbial biomass (SIR) Basal respiration
qCO2 Cmic/Corg Total N Total
P Soil organic carbon 1.00
Microbial biomass (CFM) 0.60 1.00
Microbial biomass (SIR) 0.45 -0.44 1.00
Basal respiration 0.69 0.31 0.62 1.00
qCO2 0.03 -0.61 0.20 0.55 1.00
Cmic/Corg -0.88 -0.39 0.35 0.75 -0.31 1.00
Total N 0.81 0.47 0.33 0.62 0.05 -0.83 1.00
Total P 0.61 0.48 0.45 0.35 0.35 -0.64 0.50 1.00
biomass carbon at 0-20 cm was lower than that for the Haatso series at the same depth even though the difference in the treatments however was not significant (Table 2). At 20-40 cm soil depth, the Adenta series recorded significantly higher (p<0.01) microbial biomass than that of the Haatso series. Similar observation was made for 40-60 cm, soil depth for both series. Values obtained for the average microbial biomass carbon in the topsoil compared favourably with that reported by other workers Ros et al. (2006); Kiose et al. (2004). Variation in microbial biomass carbon down the soil profile has been observed by Brammer (1972) and Thompson and Troeh (1978) that as organic matter decreased down the profile, the microbial biomass also decreased.
Microbial biomass carbon as measured by substrate induced respiration increased with soil depth (Table 2). At 0-20 cm of depth, the Adenta series recorded almost the same value as the Haatso series. For the depths 20-40 cm and 40-60cm, no significant difference was observed between the SIR values for the two soil series. The highest SIR values were recorded for 40-60 cm depth for both soil series.
The basal respiration decreased with soil depth. The basal respiration for Adenta series (0-20 cm) was not significantly higher than that of the Haatso series for the same depth (Table 2). A similar observation was made for the depths of 20-40 cm and 40-60 cm. The basal respiration is an index of potential carbon dioxide loss (Franzluebers and Arshad, 1996), it can also indicate soil quality since soil quality is presumed to be high if the basal respiration is high and vice versa. The basal respiration is also considered to reflect the availability of carbon for microbial maintenance and is a measure of the basic turnover rates in soil (Insam et al. 1991).
The qCO2 values generally decreased with depth in
both soil series. Generally, the Haatso series had similar qCO2 at the different depths as the Adenta series (Table
2) even though the qCO2 at 20 – 40 cm depth for the
Haatso series was slightly higher than the rest of the values. Increases in qCO2 are often connected to stress
either by increasing the maintenance energy of the prevalent community or by a change in the community structure. Generally the qCO2 decreases in more stable
ecosystems (Insam and Domsch, 1988) while any type of disturbance increased the qCO2 ratio (Fritz et al.
1996).The higher qCO2 for the Haatso series at the depth
of 20-40 cm could suggest shift in the microbial community structure (Bath et al., 1992) which is reflected in the low microbial biomass value and the low organic carbon attained.
The Cmic/Corg (microbial quotient) values indicate the proportion of the microbial biomass carbon in soil organic carbon. The Adenta series had a microbial quotient ranging from 4.5 to 9.00 % whilst that of the Haatso series ranged from 9.6 to 16.60 %. Even though the Haatso series had a lower soil organic carbon content, it could support a higher microbial quotient as compared to the Adenta series. This support of higher microbial quotient can be transient and it might be due to the presence of other nutrients in the environment. In the Adenta series from 0-20 cm and 20-40 cm depths the values fell within the expected ranges of 1-5 % (Jenkinson and Ladd, 1981). For the Haatso series, the values were higher than that of the Adenta series at all depths (Table 2).
Correlation between soil organic matter and microbial biomass by chloroform fumigation method (CFM) shows an r of 0.60 which is significant at p≤ 0.05 (Table 3). Correlation between microbial biomass by CFM and SIR was -0.44 (Table 3). The correlation between soil organic carbon and Cmic/Corg is -0.88 indicating that as soil organic carbon increases, less carbon goes to the microbial biomass pool, probably the carbon is partitioned to other carbon pools in the soil. Also, the total N of the soil increases with increase in soil organic carbon (r= 0.81).
CONCLUSION
Microbial biomass was determined by two methods for two soil series namely the Adenta and the Haatso series. Microbial biomass carbon (CFM) and basal respiration were generally high in the Adenta series than in the Haatso series indicating higher inherent fertility in the former soil than the latter soil. Even though no good relation was found between the two methods used in biomass determination, other parameters such as qCO2
and Cmic/Corg can aid in assessing the state of the microbial community and soil quality.
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