Phosphorus requirements of microbial biomass in a regosol and an andosol

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Soil Biol. Biochem. Vol. 30, No. 7, pp. 865±872, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(98)00011-X 0038-0717/98 $19.00 + 0.00

PHOSPHORUS REQUIREMENTS OF MICROBIAL BIOMASS IN A REGOSOL AND AN ANDOSOL HASTA PRATOPO LUKITO, KENJI KOUNO* and TADAO ANDO Faculty of Applied Biological Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima, 739 Japan (Accepted 10 November 1997) SummaryÐThe critical P concentration in microbial biomass (de®ned as that required to achieve 80% of the maximum synthesis of microbial biomass C) and minimum amount of available P to obtain the critical P concentration in the microbial biomass of a granitic regosol and an andosol of Japan were examined. Phosphorus was applied as KH2PO4 at rates of 0, 25, 50, 200 and 400 mg P kgÿ1 soil to a regosol and 0, 25, 100, 400 and 800 mg P kgÿ1 to an andosol together with 2000 mg C (rice straw) and 200 mg N (ammonium sulphate) kgÿ1. With increasing P application, the available P in soil markedly increased in the regosol and gradually increased in the andosol. The amount of microbial biomass C and P increased with available P up to 76 and 29 mg P kgÿ1 in the regosol and andosol, respectively, and either remained constant or was slightly decreased at a higher available P value. The concentration of P in the microbial biomass was higher in the regosol (29 to 89 mg P gÿ1) than in the andosol (13 to 32 mg P gÿ1), assuming that 1 g of dry biomass contained 0.5 g C. The microbial biomass C to P ratio was higher in the andosol (16 to 38) than in the regosol (6 to 17). The critical P concentration in microbial biomass was estimated to be 62 mg P gÿ1 biomass in the regosol and 19 mg P gÿ1 in the andosol. The corresponding minimum value of available P in soil to increase microbial biomass was estimated as 38 and 6 mg P kgÿ1 soil in the regosol and andosol, respectively. The speci®c respiration of microbial biomass was also very high at those P concentrations which were considered optimum in both soils to increase not only the amount of microbial biomass C and P but also microbial activity. These were 38 mg P kgÿ1 soil in the regosol and 6 mg P kgÿ1 soil in the andosol. # 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

The microbial biomass is of importance in soil nutrient cycling and in plant nutrition (Barber and Rovira, 1975; Cosgrove, 1977). The size and turnover of soil microbial biomass in¯uence the nutrient supply to plants (Anderson and Domsch, 1980; Marumoto, 1984; SchnuÈrer and Rosswall, 1987). N and P in the microbial biomass are key nutrients for plant production on granitic regosol and andosol which are widely distributed in Japanese arable land, and have very low chemical fertilities. We examined the changes in the microbial biomass P dynamics in relation to soil properties. Regosols have a coarse texture, extremely low CEC, base saturation, organic matter and available P, whereas, andosols have excellent soil structure, porosity, good water holding capacity and permeability. They are rich in allophanes and humus which contain active aluminium and highly humi®ed black colored humic acid, and have high phosphate retention and acidity. In these soils, it is important to generate the microbial biomass to improve plant production. Soil C is correlated with microbial biomass C and is the key factor for biomass formation in *Author for correspondence. E-mail: [email protected] 865

many ®elds (Sparling and West, 1988; Anderson and Domsch, 1989). Under limited C content, P status in soil has little e€ect on biomass (Brookes et al., 1982). Gallardo and Schlesinger (1994) found that microbial biomass was signi®cantly correlated with soil organic C, but not with total soil N in warm temperate forests. When organic substrate (e.g. rice straw, manure) were applied to soil in sucient amounts, N and P were also essential for synthesis of new soil microbial biomass, especially in regosols and andosols. Wardle (1992) found that the proportion of soil organic C and N immobilized in microbial biomass was related to soil N concentration. Scheu (1990) also suggested that P supply from soil limited the microbial biomass in some forest ecosystems. Chauhan et al. (1981) investigated the relationships between microbial uptake of P and available P status and organic C addition and found that the correlation between microbial P uptake and solution P values was signi®cant, and microbial C-to-P ratios ranged from 12-to-1 under high available P conditions to 45-to-1 where P was in low supply. However, the P requirement of the soil microbial biomass is still not well known. Furthermore, the critical P concentration in the microbial biomass (de®ned as that required to achieve 80% of the maximum synthesis of microbial biomass C) has

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not yet been estimated and very little information exists concerning the minimum available P requirement and the size of the microbial biomass P particularly in regosols and andosols in Japan. We examined the e€ects of P application on microbial biomass C and P formation and speci®c respiration, and estimated the critical P concentration in the microbial biomass and the minimum amount of available P in soils to obtain the critical P concentration in the microbial biomass in a regosol and an andosol following rice straw and inorganic P additions.

MATERIALS AND METHODS

Soils Two di€erent type of soils, regosol and andosol were sampled from Fukuyama and Miyoshi areas, respectively, in Hiroshima Prefecture, Japan. Chemical and physical properties of the soils and rice straw are presented in Table 1. The soil samples were collected from the upper 0±20 cm of the pro®le. After removing large pieces of plant material by hand, the soil was sieved (<2 mm) and adjusted to 40% of WHC with distilled water. The soil was then conditioned at 258C for 10 d to allow respiration to stabilize after sieving, and then used. Treatments and experimental design All soil samples were mixed with organic C: 2000 mg C (rice straw) and mineral nutrients: 200 mg N ((NH4)2SO4), 60 mg K (K2SO4), 100 mg and 40 mg Mg Ca (CaCl22H2O) (MgSO47H2O) kgÿ1 soil. Phosphorus was applied as KH2PO4 at rates of 0, 25, 50, 200 and 400 mg P kgÿ1 soil to the regosol and 0, 25, 100, 400 and 800 mg P kgÿ1 soil to the andosol. The soil pH was adjusted to 6.5 with CaCO3. The soil samples were readjusted to 40% of WHC and each weight into 100 ml glass bottle. The bottles were then placed in 1 L jars and incubated at 258C for 30 d. To trap CO2 evolved by soil microorganisms during incubation, 20 ml of 1 M NaOH solution was placed inside each jar. Soil samples were divided into two portions, one was used for microbial analyses and another portion was air-dried and used for chemical analyses. Microbial biomass C, microbial biomass P, microbial speci®c respiration and 0.5 M NaHCO3-extractable P in soils were measured at 5, 10 and 30 d after P application.

Microbial biomass measurement Microial biomass C was determined by the fumigation extraction (FE) method (Wu et al., 1990). Moist soil samples containing 20 and 15 g dry soil of regosol and andosol, respectively, were fumigated with ethanol-free CHCl3 for 24 h. After CHCl3 removal, the soil samples were extracted with 80 and 60 ml of 0.5 M K2SO4 for regosol and andosol, respectively. The organic C extracted was analyzed by a total organic carbon analyzer (Shimadzu TOC5000). Microbial biomass C was estimated from the increase in K2SO4-extractable organic C (EC) after fumigation, where EC is organic C extracted in the fumigated samples minus organic C extracted in the non-fumigated samples. Microbial C (BC) was estimated from the equation: BC=2.22  EC. Microbial biomass P in the regosol was determined by the FE method of Brookes et al. (1982), using 10 g of moist sample. Microbial biomass P was calculated as sodium bicarbonate-extractable inorganic P in fumigated soil minus that extracted from unfumigated soil then divided by a KP value of 0.40, assuming that 40% of P in the microbial biomass is released as inorganic P by CHCl3. Microbial biomass P was further corrected for P ®xation during bicarbonate extraction by measuring the recovery of added KH2PO4 (equivalent to 25 mg P kgÿ1). Microbial biomass P in the andosol was determined by the anion exchange membrane (AEM) technique developed by Kouno et al. (1995). This method was developed to overcome the marked P ®xation by these soils during extraction and interference in the colorimetric assay for P by the dark color of extracted humic substances. Again moist soil was used (equivalent to 1.5 g oven-dry soil). Microbial biomass P was further corrected by a similar method using the equation: microbial P = EP/KP100/R, where EP=(P released by CHCl3 and extracted by distilled water) minus (P released from non-fumigated soil), KP=0.40 at 258C, R = percentage recovery of added P as KH2PO4. Microbial speci®c respiration Microbial speci®c respiration was measured as CO2 evolution from soil samples which was trapped in NaOH. During subsequent incubation, NaOH was renewed every 5 d. Total CO2 was then titrated with 0.4 M HCl using an automatic titrator. The microbial speci®c respiration was expressed as mg CO2±C evolved per unit microbial biomass C (mg mic.C) and unit time (d).

Table 1. Chemical and physical properties of the regosol, andosol and rice straw used Material used Regosol Andosol Rice straw

Texture

pH 1:2.5 N KCl

Organic C (mg gÿ1)

Total N (mg gÿ1)

Total P (mg P gÿ1)

Available P (Olsen) (mg P gÿ1)

SL L ND*

4 4.1 ND

4.6 83.4 345

0.2 3.7 5.7

172 380 692

2.95 1.02 ND

*Not determined.

P retention capacity Bulk density (mg P gÿ1) (g cmÿ3) 1.49 10.26 ND

1.31 0.65 ND

Critical P concentration of microbial biomass

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Soil chemical and physical analysis

Statistics

Total C and total N in the air-dried samples were determined using a carbon and nitrogen analyzer (Yanaco C-N Corder MT 500). Total P in soil was extracted by H2SO4 and HNO3 (1:1, v/v) doubleacid digestion and determined colorimetrically by the ammonium-molybdate ascorbic acid method (Murphy and Riley, 1962). Phosphate retention capacity of soils was estimated from the amount of P absorbed by 50 g soil from 100 ml of 25 g lÿ1 diammonium hydrogen phosphate (5863 mg P lÿ1, pH 7.0) for 24 h and expressed as mg P absorbed gÿ1 soil (Nanjyo, 1986). Available P was determined in aliquots of 0.5 M NaHCO3 (pH 8.5) by the ammonium molybdate blue method (Olsen et al., 1954). Soil pH was measured using a glass electrode (1:2.5 soil±N KCl). WHC was determined as described by Jenkinson and Powlson (1976). Soil bulk density was determined by the core method proposed by Blake and Hartge (1986).

All results are expressed as a mean of three replicate determinations on an oven dry (1058C, 24 h) soil basis. And a one-way ANOVA was performed to determine the signi®cant di€erences between treatments and sampling dates, followed by test on LSD. RESULTS AND DISCUSSION

E€ects of P application on microbial biomass C and P formation E€ects of P application on microbial biomass C and P in regosol and andosol soils are presented in Fig. 1. In regosol [Fig. 1(A), (C)] the amount of microbial biomass C and P increased with P application up to 200 mg P kgÿ1 and then remained constant. The microbial biomass C and P were maximum 10 days after treatment. With P application, the amount of microbial biomass C in regosol increased gradually to the maximum amount (620 mg C kgÿ1 soil), while microbial biomass P

Fig. 1. Microbial biomass C and P at 5 (R), 10 (*) and 30 (Q) d after P application to the regosol (A, C) and the andosol (B, D). Each value is the mean of three replications, bars indicate LSD < 0.05.

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without addition of dissolved P did not increase the amount of resin bicarbonate-extractable P or microbial biomass P. Chauhan et al. (1979) found that glucose caused microbial depletion of available P at minimum values of P availability. Concentration of P and C-to-P ratio in microbial biomass

Fig. 2. E€ects of P application rate on available P in the regosol (Q) and the andosol (*). Each value is the mean of three replications, bars indicate LSD < 0.05.

increased rapidly to the maximum amount (90 mg P kgÿ1 soil). Similarly, the amount of microbial biomass C and P in the andosol [Fig. 1(B), (D)] were highest at 10 d irrespective of P amount and increased with P application up to 400 mg kgÿ1 and remained constant at higher rates. The maximum microbial biomass C and P in the andosol were 1108 and 48 mg kgÿ1 soil, respectively. Available P concentration in soil increased markedly in the regosol and slightly in the andosol with P application (Fig. 2). Although the application of 200 mg P resulted in 100 mg available P kgÿ1 in the regosol, 400 mg P application resulted in only 28 mg available P kgÿ1 in the andosol. In general, andosols are highly acidic, contain large amounts of free Al and have a high phosphate absorption coecient (Kanazawa et al., 1988). The low available P value in the andosol may have been caused by the high P ®xation capacity of this soil. The growth of microbial biomass is markedly controlled by the amount of available organic C (Marumoto, 1984; Powlson et al., 1987). We added the same quantity of rice straw to both soils, and the amount of microbial biomass C and P increased with available P concentration up to 76 mg kgÿ1 in the regosol and 29 mg kgÿ1 in the andosol [Fig. 3(A), (C)]. Above this available P value, microbial biomass C and P remained constant or decreased slightly. The smaller amount of microbial biomass P in the andosol resulted from the smaller amount of available P in this soil. Gallardo and Schlesinger (1994) also suggested that microbial biomass in the mineral soil is limited by the P availability. The amount of microbial biomass C in the andosol was larger than that in the regosol. This may be due to the di€erence in bulk density, which was lower in the andosol (0.65) than in the regosol (1.31) (Table 1). According to unit soil volume [Fig. 3(B), (D)], the amount of microbial biomass C in the andosol resembled that in the regosol, depending on the available P value. Lee et al. (1990) found that the presence of carbon in soil

The concentration of P in the microbial biomass (assuming that 1 g of dry biomass contains 0.5 g C) was higher in the regosol than in the andosol (Table 2). Concentration of P in microbial biomass was between 29 and 89 mg P gÿ1 in the regosol, and between 13 to 32 mg P gÿ1 in the andosol. The values for the andosol are in agreement with those reported by Brookes et al. (1984), Srivastava and Singh (1988), and Singh and Singh (1995), but those for the regosol were higher. Although the reason for these di€erences is not clear, we used controlled incubation condition while they measured the microbial biomass in the ®eld (natural conditions) which might be a€ected by plant P uptake, environmental conditions and soil management practices. Many microorganisms grown in vitro are able to accumulate more P in the form of polyphosphates than necessary for growth (Harold, 1966). Furthermore, Van Veen and Paul (1979) reported that the concentration of P in microorganisms grown in vitro can vary widely, depending on the growth conditions. The microbial biomass C-to-P ratio in the andosol was consistently higher than that in the regosol (Table 2). The microbial biomass C-to-P ratio ranged from 5.6 to 17.3 in the regosol and from 15.7 to 37.5 in the andosol. The higher microbial biomass C-to-P ratio and low P concentration in the biomass in the andosol may have been caused by competitive absorption between microbial biomass and soil colloids or by the di€erence in the micro¯ora of the two soils, whereas, the lower microbial C-to-P ratio and high P concentration in the microbial biomass in the regosol by excess P absorption in microbial biomass. The microbial C-to-P ratio of the regosol was comparable with those reported by William and Sparling (1984) and Joergensen et al. (1995), whereas, that for the andosol was similar to those reported by Brookes et al. (1984), Sarathchandra et al. (1984), Srivastava (1992) and Singh and Singh (1995). Critical level of P concentration in microbial biomass The critical nutrient concentration (Ulrich, 1952) which has been used extensively for diagnosis of plant nutrient de®ciencies has been shown to vary with plant part sampled and plant age (Bailey et al., 1983). Furthermore, expression of critical nutrients on a fresh weight basis may reduce variation because of the changing dry matter content of plants with age. The critical amount of P in leaves

Critical P concentration of microbial biomass

869

Fig. 3. E€ect of available P on microbial biomass C (A, B) and P (C, D) in the regosol (Q) and the andosol (*). A and C calculated by microbial biomass per soil weight (mg P gÿ1), B and D calculated by microbial biomass per soil volume (mg P cmÿ3). Each value is the mean of three replications, bars indicate LSD < 0.05.

which has been reported for ryegrass, wheat, and bean was estimated by the relative dry matter yield to achieve 80% of the maximum yield (Fohse et al., 1988). The critical P value in the microbial biomass was estimated from the relative amount of biomass to achieve 80% of the maximum synthesis of microbial biomass C, where, dry matter yield was considered as the amount of microbial biomass C while

the P concentration in dry matter was considered as P concentration in the microbial biomass. For each soil, relative microbial biomass C to the maximum value of each sampling time (5, 10, 30 d after treatment) was plotted on the ordinate (Y-axis) against concentration of P in the microbial biomass on the abscissa (X-axis) in Fig. 4. The P concentration corresponding to the arbitrary point at 80% to achieve

Table 2. Concentration of P in microbial biomass and microbial biomass C-to-P ratio Concentration of P in microbial biomass (mg gÿ1)*

Applied P (mg P kgÿ1 soil) 0 25 50 100 200 400 800

Microbial biomass C-to-P ratio

regosol incubation period (d)

andosol incubation period (d)

regosol incubation period (d)

andosol incubation period (d)

5

5

5

10

30

5

10

30

11.3 7.7 6.5

17.3 11.2 9.5

15.9 9.7 8.6

29.3 17.7

37.5 36.0

25.6 20.4

5.7 5.6

7.1 7.1

6.9 6.8

19.5

33.1

24.5

15.9 15.7

23.2 21.1

17.6 15.8

10

30

44 65 77

29 45 52

31 51 58

88 89

71 71

72 73

10

30

17 28

13 14

19 25

26

15

20

31 32

22 24

28 32

Assuming that dry biomass contains 50% C (Brookes et al., 1984).

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Fig. 4. Relationship between relative value of microbial biomass C to the maximum value of each sampling time and concentration of P in microbial biomass in the regosol (A) and the andosol (B). After 5, 10 and 30 d of incubation. Each value is the mean of three replications.

the maximum amount of microbial biomass C was estimated by the ®tted curve. Since the P concentration in the biomass is the average concentration of many organisms, we did not determine the absolute critical P concentration in the biomass. However, we could estimate the approximate critical concentration by using the relative value of biomass C against the maximum biomass C at each sampling time. The critical P concentration in the microbial biomass to achieve 80% of the maximum microbial biomass C was higher in the regosol (62 2 4.7 mg P gÿ1) than in the andosol (19 2 4.2 mg P gÿ1). The approximate critical value of P obtained on some tropical pasture legumes (Andrew and Robins, 1969) was 2.2±2.4 mg P gÿ1. De Wit et al. (1963) reported that temperate grass in the vegetative stage has a critical P value of 2.2 mg P gÿ1, and Lunt et al. (1965) found 3.5 mg P gÿ1 in grass regrowth. Since the critical values of P in microbial biomass in the regosol and andosol were 62 2 4.7 and 19 2 4.2 mg P gÿ1, respectively, the critical P concentration in the microbial biomass was markedly higher than those in leaves or shoots in plants. Minimum amount of available P in soil to obtain the critical P concentration in the microbial biomass Microbial biomass was signi®cantly correlated with available (NaHCO3 extractable) P in soils, as reported in an arid tropical soil (Srivastava, 1992) and a mineral soil (Gallardo and Schlesinger, 1994). To determine the minimum amount of available (NaHCO3 extractable) P in soil to obtain the critical P concentration in the microbial biomass, the available P was plotted on the X axis against the

concentration of P in the microbial biomass on the Y axis. When the P concentration in microbial biomass was achieved at 62 2 4.7 mg P gÿ1 in the regosol [Fig. 5(A)] and 19 2 4.2 mg P gÿ1 in the andosol [Fig. 5(B)] which were critical P levels in biomass, the corresponding available P in soils were 38 2 1.6 and 6 2 0.9 mg P kgÿ1 for the regosol and the andosol, respectively. The minimum amount of available P in the andosol was lower than that in the regosol. These di€erences might be caused by the micro¯ora or by excessive P absorption into microbial biomass in the regosol. According to simple regression analyses, microbial biomass C was correlated with microbial biomass P with the equation Y = 240.57 + 4.27X (R2=0.997, P < 0.01) for the regosol and Y = 515.87 + 12.02X (R2=0.736, P < 0.01) for the andosol. The variability in microbial biomass C has been reported to account for 78% (Srivastava and Singh, 1988) and 81% (Brookes et al., 1984) of the variance to that in microbial biomass P. The evaluation of the minimum amount of available P to increase the microbial biomass C in the regosol and the andosol therefore, could be used as the estimation of the amount needed to increase the microbial biomass P in these soils. According to the standards of grassland management in Japan, at least 10 mg available-P kgÿ1 soil is required for grasses (Egawa and Sekiya, 1962). The minimum amount of available P to increase microbial biomass P was 38 2 1.6 mg P kgÿ1 in the regosol and 6 2 0.9 mg P kgÿ1 in the andosol. These ®ndings indicated that the minimum amount of available P in soils required to increase the microbial biomass was higher than that required for plant growth.

Critical P concentration of microbial biomass

871

Fig. 5. Relationship between concentration of P in microbial biomass and available P in the regosol (A) and the andosol (B) after 5, 10 and 30 d of incubation. Each value is the mean of three replications.

Our ®ndings are in agreement with the compartments and ¯ows of P suggested by Stewart and McKercher (1982). Paul and Clark (1989) explained that more of the mineralized phosphorus is used by the soil organisms than by the plants; microorganisms contain 16 to 68 mg P gÿ1, while plants contain about 2.2 to 3.5 mg P gÿ1. The relationship between available P in soils and microbial biomass activity was also examined (Fig. 6). The speci®c respiration of the microbial biomass (called metabolic quotient of CO2 by

Anderson and Domsch, 1985) is calculated as the ratio of CO2±C evolved during 30 d of incubation per unit of microbial biomass per day. In our study, the microbial activity of the regosol and the andosol markedly increased with the available P in soil up to 33 and 6 mg P kgÿ1, respectively, and then decreased. The microbial activity was also very high at 30±40 mg P for the regosol and 6±7 mg P kgÿ1 for the andosol during incubation. The minimum amount of available P to obtain the critical P concentration in the microbial biomass was approximately 38 and 6 mg P kgÿ1 in the regosol and the andosol, respectively. This is in agreement with the available P considered as necessary to increase the amount of microbial biomass C and P and their activity. AcknowledgementsÐThis work was supported by the grants-in-aid for Scienti®c Research from the Ministry of Education, Sciences, Sports and Culture of Japan. REFERENCES

Fig. 6. E€ects of available P on microbial biomass activity in the regosol (Q) and the andosol (*). Each value is the mean of three replication at 30 d of incubation, bar indicates LSD < 0.05.

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