Microbial biomass in soils with calcium accumulation associated with the application of composted lime-treated sewage sludge

Abstract

We investigated the factors affecting the microbial biomass in upland soils where calcium (Ca) had accumulated because of the application of composted lime-treated sewage sludge (LSS). Soil samples (0–10 cm) were taken from field plots that had received LSS compost at the rates of 0, 20, 40 and 60 Mg ha⁻¹ year⁻¹ for 4 years. The pH values and the amount of exchangeable Ca²⁺ increased with increasing amounts of compost. Total concentrations of cadmium, copper and zinc increased with the application of LSS compost, and at a rate of 60 Mg ha⁻¹ year⁻¹, they were 0.7, 41 and 144 Mg kg⁻¹ soil, respectively. Electrical conductivity (EC) increased in the soils that had received LSS compost, and this was ascribed to an increase in the amount of water-soluble Ca²⁺. The application of LSS compost led to a decrease in the ratio of microbial biomass C to total soil C and an increase in biomass specific respiration (qCO₂), indicating adverse effects of LSS compost application on soil microbial biomass, as observed in sewage sludge-amended soils. Analysis of the ergosterol content did not reveal any increase in the proportion of fungal biomass in the total microbial biomass in contrast to earlier findings on sewage sludge-amended soils. The concentrations of heavy metals in the soils used were below the levels known to exert adverse effects on soil microbial biomass. In contrast, the ratio of microbial biomass C to total soil C decreased significantly and the qCO₂ increased significantly with the increase in EC and in the amount of water-soluble Ca²⁺. Therefore, it was suggested that the electrolyte concentration associated with the amount of water-soluble Ca²⁺ was the major factor affecting microbial biomass in soils amended with LSS compost.

Key words:

• compost
• electrical conductivity
• lime-treated sewage sludge
• microbial biomass
• qCO₂

INTRODUCTION

Large amounts of sewage sludge have been produced in advanced countries. In Japan, more than 400 million m³ of sewage sludge is generated annually and most of it is disposed of by landfilling. However, a recent lack of landfill sites has promoted the effective utilization of sewage sludge. One of the alternative methods to dispose of sewage sludge is the application of the sludge to agricultural land after composting. The application of sewage sludge compost to agricultural land has been practiced in many countries. Nevertheless, there is a concern about the toxic effects of heavy metals accumulated by the application of sewage sludge on the soil microbial biomass (Brookes and McGrath 1984; Chander and Brookes 1991; Chander et al. 1995; Fließbach et al. 1994). Sewage sludge contains polymers or lime used for the coagulation process. Lime-treated sewage sludge (LSS) is relatively easy to compost, compared with polymer-treated sewage sludge. In our previous study (Aoyama et al. 2003), we reported that the application of compost made from LSS led to the accumulation of exchangeable Ca²⁺ and thus increased the soil pH. An increase in soil pH generally results in an increase in microbial biomass and microbial activities in soils (Adams and Adams 1983; Carter 1986; Soon and Arshad 2005). In contrast, the accumulation of soluble salts exerts adverse effects on soil microbial biomass (Killham 1985; Rietz and Hynes 2003; Sardinha et al. 2003).

Our objective in the present study was to investigate the factors affecting microbial biomass in upland soils where calcium (Ca) had accumulated because of the application of composted LSS. We measured microbial biomass, ergosterol content and respiration in soil samples taken from field plots that had received LSS compost at the rates of 0, 20, 40 and 60 Mg ha⁻¹ year⁻¹ for 4 years. Heavy metal concentrations, electrical conductivity (EC) and the composition of water-soluble ions were also analyzed.

MATERIALS AND METHODS

Site and soils

Field plots were established in 1998 at the Aomori Prefectural Agriculture and Forestry Research Center, Kuroishi, Japan. The soil at this site is an alluvial soil classified as a Gray Lowland soil (Udifluvent). Details of the trial have been reported by Aoyama et al. (2003). The experiment was arranged in a split-plot design with LSS compost as the main factor and fertilizer treatments as secondary factors. The compost was applied at the rates of 20, 40 and 60 Mg ha⁻¹ year⁻¹, in addition to a control (0 Mg ha⁻¹ year⁻¹). Fertilizer treatments consisted of NPK treatments at three different N fertilization rates (133, 200 and 266 kg ha⁻¹ year⁻¹) and a control (no-fertilizer treatment). The 20 Mg ha⁻¹ year⁻¹ treatment lacked the “no-fertilizer plot”. The size of each plot was 3.5 m × 10 m. Crops were cultivated twice a year as follows: cabbage (Brassica oleracea L.; first crop) and turnip (Brassica rapa L.; second crop) in 1998; cabbage (first crop) and Japanese radish (Raphanus sativus L.; second crop) in 1999 and 2000; and cabbage (first and second crops) in 2001. The LSS compost and fertilizer applications were carried out in April before the planting of the first crop every year. The composts were commercially available and made from sewage sludge treated with lime for coagulation. The chemical properties of the composts used are shown in Table 1.

Soil samples were taken from the 0–10 cm layer in July (after harvest of the first crop) and September (after harvest of the second crop) 2001. Four subsamples were taken and pooled in each plot. The soil samples were passed through a 2-mm mesh sieve. The moist subsamples were used for the analysis of microbiological properties and the residual samples were air-dried for chemical analyses.

Analysis of soil chemical properties

Soil pH values were determined using a glass electrode at a ratio of 1:2.5 in water and 1 mol L⁻¹ KCl. The total C and N contents were simultaneously determined using a Sumigraph NC-90A automatic analyzer (Sumitomo Chemical Company, Osaka, Japan). Exchangeable cations were extracted with 1 mol L⁻¹ CH₃COONH₄ (pH 7) using a soil-to-solution ratio of 1:10, and the amounts of Ca²⁺, Mg²⁺, K⁺ and Na⁺ in the extract were determined by atomic absorption spectrometry. The total concentrations of Cd, Cu and Zn in the soil samples taken in September were determined by atomic absorption spectrometry, following digestion with an HClO₄–HNO₃ mixture. The EC value was measured in a 1:5 water extract. In the same water extract, the amounts of water-soluble cations (Ca²⁺, Mg²⁺, K⁺ and Na⁺) were determined by atomic absorption spectrometry and the amounts of water-soluble anions (Cl⁻, NO⁻ ₃ and SO²⁻ ₄) by indirect photometric ion chromatography (Aoyama 1992).

Analysis of soil microbial properties

For the analyses of microbial biomass C (C_mic) and ergosterol content, moist soil samples equivalent to 60 g oven-dry soil were weighed in 100 mL plastic beakers and adjusted to 60% of the maximum water-holding capacity (MWHC) with distilled water and then pre-incubated for 7 days at 25°C. The C_mic was measured using the fumigation–extraction procedure proposed by Vance et al. (1987). In brief, a portion of the pre-incubated soil sample corresponding to 25 g oven-dry soil was fumigated with alcohol-free chloroform for 24 h. Organic C was extracted from the fumigated and unfumigated soil samples with 100 mL of 0.5 mol L⁻¹ K₂SO₄ and quantified using a TOC analyzer (TOC-V_E; Shimadzu Company, Kyoto, Japan). A k _EC factor of 0.45 was used

Table 1 Chemical properties of the sewage sludge compost

Year	Moisture^† (g kg^−1)	pH^‡	EC^‡ (dS m^−1)	Total C^§ (g kg^−1)	Total N^§ (g kg^−1)	C/N ratio	Total Ca^§ (g kg^−1)	Total Cd^§ (mg kg^−1)	Total Cu^§ (mg kg^−1)	Total Zn^§ (mg kg^−1)
1998	419	7.77	3.86	201	22.8	8.8	178	3.3	354	1061
1999	479	7.64	3.72	203	21.7	9.4	74	1.8	240	861
2000	470	7.92	4.70	176	20.5	8.6	177	1.6	190	639
2001	363	8.08	3.15	159	16.6	9.6	194	3.1	150	686
Average	433	7.85	3.86	185	20.4	9.1	156	2.4	234	812
^†Wet weight basis;
^‡Measured in a 1:10 aqueous suspension;
^§Oven-dry weight basis.

Table 2 Chemical properties of soils

Amount of compost applied (Mg ha^−1 year^−1)	pH (H2O)	pH (KCl)	Total C (g kg^−1)	Total N (g kg^−1)	C/N ratio	Exchangeable bases (cmolc kg^−1)
						Ca	Mg	K	Na
July
0	5.34 ± 0.12	4.45 ± 0.10	38.6 ± 0.5	3.25 ± 0.03	11.9 ± 0.2	7.4 ± 0.7	1.54 ± 0.12	0.41 ± 0.08	0.32 ± 0.03
20	6.70 ± 0.10	6.14 ± 0.04	42.3 ± 0.2	3.61 ± 0.06	11.7 ± 0.2	32.2 ± 1.4	2.27 ± 0.06	0.25 ± 0.02	0.27 ± 0.01
40	7.16 ± 0.02	6.71 ± 0.03	44.2 ± 0.8	3.84 ± 0.03	11.5 ± 0.2	33.9 ± 0.2	2.34 ± 0.11	0.31 ± 0.08	0.27 ± 0.02
60	7.36 ± 0.04	7.03 ± 0.02	50.9 ± 0.9	4.42 ± 0.17	11.5 ± 0.2	45.1 ± 1.1	2.87 ± 0.12	0.25 ± 0.05	0.28 ± 0.03
Sept.
0	5.35 ± 0.09	4.52 ± 0.08	38.6 ± 0.8	3.21 ± 0.07	12.0 ± 0.2	10.4 ± 0.6	1.80 ± 0.10	0.39 ± 0.10	0.38 ± 0.05
20	6.77 ± 0.07	6.14 ± 0.07	41.9 ± 0.7	3.33 ± 0.04	12.6 ± 0.1	27.3 ± 0.4	2.15 ± 0.09	0.24 ± 0.05	0.26 ± 0.04
40	7.30 ± 0.08	6.80 ± 0.05	44.2 ± 0.2	3.62 ± 0.07	12.2 ± 0.3	41.8 ± 0.5	2.27 ± 0.05	0.21 ± 0.03	0.22 ± 0.01
60	7.61 ± 0.05	7.05 ± 0.03	47.5 ± 0.6	3.92 ± 0.10	12.1 ± 0.2	50.7 ± 0.9	2.59 ± 0.04	0.24 ± 0.03	0.24 ± 0.02
LSD	0.24	0.18	2.1	0.29	0.6	2.4	0.29	0.20	0.08
Values represent means ± standard error.

Table 3 Heavy metal concentrations of soils sampled in September

Amount of compost applied (Mg ha^−1 year^−1)	Cd (mg kg^−1)	Cu (mg kg^−1)	Zn (mg kg^−1)
0	0.34 ± 0.03	30.2 ± 0.2	91.4 ± 1.8
20	0.50 ± 0.06	32.5 ± 0.6	109.8 ± 0.4
40	0.47 ± 0.07	36.8 ± 0.8	126.1 ± 1.6
60	0.66 ± 0.04	41.4 ± 0.4	143.9 ± 3.0
LSD	0.17	1.7	6.6
Values represent means ± standard error.

to estimate the C_mic from extractable C (Wu et al. 1990). Ergosterol was extracted from each pre-incubated soil sample (4 g moist soil) with methanol (6 mL) by physical disruption and quantified using high performance liquid chromatography (HPLC) (Gong et al. 2001). The HPLC analysis was carried out using a Superiorex Octadecyl Silane (ODS) column (4.6 mm internal diameter × 250 mm; Shiseido Company, Tokyo, Japan) and methanol as the eluent, and the effluent was monitored by the absorbance at 282 nm.

For the measurement of soil respiration, moist soil samples equivalent to 30 g oven-dry soil were weighed in 50 mL glass vials and then adjusted to 60% of the MWHC with distilled water. After pre-incubation for 7 days at 25°C, each vial was placed in a 900 mL airtight glass jar containing 5 mL of water to maintain a moist atmosphere and a plastic vial with 5 mL of 1 mol L⁻¹ NaOH to trap CO₂, and incubated for 7 days at 25°C (Aoyama and Nagumo 1997). The amount of CO₂ in NaOH was determined by titration with 0.1 mol L⁻¹ HCl using an automatic titrator.

Statistics

Data for the soil chemical and microbial properties were analyzed using two-way anovas (Yanai 1998). For all data, the effects of the fertilizer treatments were not statistically significant at P = 0.05. Thus, means for each compost application rate were separated by the least significant difference (LSD) test at P = 0.05 (Yanai 1998).

RESULTS AND DISCUSSION

Effects on soil chemical properties

The pH values in H₂O and KCl were considerably lower in the soil without LSS compost application (Table 2). Both soil pH values increased significantly with increasing amounts of LSS compost. Both soil pH values exceeded 7 in the 60 Mg ha⁻¹ year⁻¹ treatment. The amount of exchangeable Ca²⁺ also increased with the application of LSS compost, reflecting the high concentration of Ca in the compost (Table 1). Thus, the increase in soil pH could be attributed to the accumulation of exchangeable Ca²⁺ derived from the LSS compost.

The application of LSS compost increased significantly the total C and N contents, demonstrating the accumulation of organic matter derived from LSS compost. However, the C/N ratio was not significantly affected by LSS compost application.

The concentrations of Cd, Cu and Zn in the soils sampled in September increased significantly with increasing amounts of LSS compost (Table 3). Nevertheless, the concentrations were relatively low, even in the 60 Mg ha⁻¹ year⁻¹ treatment.

Overall, the EC value was higher in September than in July (Table 4). The application of LSS compost

Table 4 Electrical conductivity (EC) and water-soluble ion contents in soils

Amount of compost applied (Mg ha^−1 year^−1)	EC (dS m^−1)	Water-soluble cations (cmolc kg^−1)					Water-soluble anions (cmolc kg^−1)
		Ca^2+	Mg^2+	K^+	Na^+	Total	Cl^−	NO^− 3	SO^2− 4	Total
July
0	0.10 ± 0.01	0.18 ± 0.03	0.10 ± 0.01	0.028 ± 0.009	0.090 ± 0.005	0.40 ± 0.05	0.03 ± 0.00	0.12 ± 0.06	0.17 ± 0.03	0.32 ± 0.05
20	0.32 ± 0.09	0.90 ± 0.23	0.19 ± 0.04	0.012 ± 0.002	0.062 ± 0.005	1.16 ± 0.27	0.23 ± 0.08	0.26 ± 0.17	0.46 ± 0.08	0.95 ± 0.32
40	0.46 ± 0.02	1.56 ± 0.06	0.24 ± 0.01	0.013 ± 0.004	0.059 ± 0.007	1.87 ± 0.08	0.54 ± 0.08	0.57 ± 0.07	0.66 ± 0.03	1.77 ± 0.16
60	0.85 ± 0.16	2.77 ± 0.53	0.43 ± 0.08	0.011 ± 0.003	0.060 ± 0.010	3.27 ± 0.63	1.16 ± 0.18	1.18 ± 0.42	1.04 ± 0.15	3.39 ± 0.75
Sept.
0	0.12 ± 0.01	0.25 ± 0. 50	0.13 ± 0.02	0.026 ± 0.013	0.102 ± 0.022	0.51 ± 0.09	0.04 ± 0.00	0.03 ± 0.01	0.29 ± 0.06	0.36 ± 0.06
20	0.26 ± 0.03	1.08 ± 0.05	0.20 ± 0.02	0.008 ± 0.004	0.060 ± 0.013	1.34 ± 0.07	0.07 ± 0.01	0.05 ± 0.01	0.65 ± 0.03	0.78 ± 0.04
40	0.28 ± 0.03	1.21 ± 0.11	0.16 ± 0.01	0.006 ± 0.001	0.045 ± 0.010	1.42 ± 0.13	0.23 ± 0.03	0.17 ± 0.05	0.64 ± 0.10	1.04 ± 0.13
60	0.33 ± 0.04	1.28 ± 0.13	0.17 ± 0.01	0.006 ± 0.002	0.041 ± 0.002	1.50 ± 0.14	0.27 ± 0.03	0.22 ± 0.06	0.60 ± 0.10	1.09 ± 0.14
LSD	0.21	0.69	0.11	0.020	0.034	0.82	0.25	0.53	0.27	0.97
Values represent means ± standard error.

Figure 1  Relationships between electrical conductivity (EC) values and the amounts of water-soluble cations and anions. ***P < 0.001.

increased the EC regardless of sampling time. The amounts of water-soluble cations and anions also increased significantly with the application of LSS compost. In the LSS compost treatments, Ca²⁺ was the predominant water-soluble cation and its amount increased markedly with an increase in the amount of compost applied, presumably because of the accumulation of Ca induced by LSS compost application. For the water-soluble anions, Cl⁻, NO⁻ ₃ and SO²⁻ ₄ showed similar trends of increasing amounts with an increase in the rate of compost application. Both the total amounts of water-soluble cations and anions were linearly correlated with EC (Fig. 1), indicating that EC depended on the concentrations of the water-soluble ions.

Effects on soil microbial properties

In July, compared with the unamended treatment, LSS compost application did not significantly affect the amount of C_mic in the 20 and 40 Mg ha⁻¹ year⁻¹ treatments, but decreased it significantly in the 60 Mg ha⁻¹ year⁻¹ treatment (Fig. 2a). In September C_mic significantly increased in the 20 Mg ha⁻¹ year⁻¹ treatment and significantly decreased in the 40 and 60 Mg ha⁻¹ year⁻¹ treatments. Chander and Brookes (1991) and Fließbach et al. (1994) considered that the ratio of C_mic to soil organic C was a useful indicator for evaluating the effects of heavy metals on the microbial biomass in soils amended with sewage sludge. When the ratio of microbial C to total soil C (C_mic/T − C) was calculated, it decreased significantly with an increase in the amount of LSS compost in

Figure 2  Amounts of microbial biomass C (Cmic) expressed on (a) an oven-dry soil basis and (b) on a total soil C basis in the soils sampled in July (□) and September (). Error bars represent standard error. Bars with the same letter do not differ significantly according to Fisher's protected least significant difference (P = 0.05).

both July and September (Fig. 2b). This result suggests that the application of LSS compost adversely affects soil microbial biomass.

Ergosterol is peculiar to fungal cells and is closely related to the fungal biomass in soil (Ruzicka et al. 2000; Stahl and Parkin 1996). In July there were no significant differences in the ergosterol content among the treatments (Fig. 3a). In contrast, the ergosterol content was significantly reduced by the application of LSS compost in September. When the ratio of ergosterol to C_mic (ergosterol/C_mic) was calculated, the results showed the same trend as that for ergosterol (Fig. 3b). Thus, significant seasonal fluctuations in the proportion of fungal biomass in the total microbial biomass were revealed for the LSS compost treatments.

The application of LSS compost not only increased soil respiration but also biomass specific respiration (qCO₂), regardless of sampling time (Fig. 4). Killham (1985) observed that the ratio of respired C to biomass-incorporated C increased with increasing magnitude of environmental stresses, such as heavy metal pollution, acid rain and salinity, when ¹⁴C-labeled glucose was added to the soils. He ascribed the increase in the ratio caused by the environmental stresses to the increase in the diversion of carbon from biosynthesis to maintenance energy requirements. This mechanism has been adopted to explain the high qCO₂ in heavy metal-polluted soils (Brookes 1995). Therefore, in addition to the decrease in the C_mic/T − C ratio, the increase in qCO₂ indicates the adverse effects of LSS compost on soil microbial biomass.

Factors affecting the microbial biomass

In the soils amended with sewage sludge, the ratio of C_mic to soil organic C decreased, whereas qCO₂ increased (Chander and Brookes 1991; Fließbach et al. 1994; Valsecchi et al. 1995). These phenomena induced by sewage sludge application have been ascribed to the accumulation of heavy metals in the soil (Giller et al. 1998). In the present study, the application of LSS compost also resulted in a decrease in the ratio of C_mic/T − C and in an increase in the qCO₂.

The concentrations of Cd, Cu and Zn in the soil samples with 60 Mg ha⁻¹ year⁻¹ of LSS compost were 0.7, 41 and 144 mg kg⁻¹ soil, respectively. In sewage-sludge-amended soils polluted with similar levels of Cd, Cu and Zn, a decrease in the ratio of C_mic to soil organic C and an increase in the qCO₂ were not observed (Fließbach et al. 1994). Chander et al. (1995), who analyzed the C_mic in soils incubated with heavy metal-enriched sewage sludge, reported that individual heavy metals did not exert harmful effects on the C_mic at a concentration of 3 mg Zn kg⁻¹ soil, 150 mg Cu kg⁻¹ soil or 300 mg Zn kg⁻¹ soil. Rost et al. (2001) showed that C_mic did not decrease at 467 mg Zn kg⁻¹ soil, and further that the

Figure 3  Amounts of ergosterol expressed on (a) an oven-dry soil basis and (b) on a total soil C basis in the soils sampled in July (□) and September (). Error bars represent standard error. Bars with the same letter do not differ significantly according to Fisher's protected least significant difference (P = 0.05).

Figure 4  (a) Soil respiration and (b) biomass specific respiration (qCO2) in the soils sampled in July (□) and September (). Error bars represent standard error. Bars with the same letter do not differ significantly according to Fisher's protected least significant difference (P = 0.05).

Figure 5  Relationships between (a) electrical conductivity (EC) and the ratio of microbial C to total soil C (Cmic/T-C), (b) between EC and biomass specific respiration (qCO2), (c) between the amount of water-soluble Ca2+ and Cmic/T-C ratio and (d) between the amount of water-soluble Ca2+ and qCO2. ***P < 0.001.

qCO₂ did not increase at 867 mg Zn kg⁻¹ soil. These findings indicated that the adverse effects of the application of LSS compost on soil microbial biomass were induced by factors other than heavy metals.

The application of LSS compost significantly increased the soil pH values. However, an increase in soil pH generally exerts favorable effects on soil microbial biomass and microbial activities (Adams and Adams 1983; Carter 1986; Soon and Arshad 2005). In contrast, soil microbial biomass and its activities are influenced by soil salinity (Killham 1985). In the present study, the application of LSS compost led to an increase in the EC and in the amounts of water-soluble ions as a consequence of the accumulation of Ca in soils. Rietz and Haynes (2003) reported that with an increase in EC, the ratio of C_mic to soil organic C decreased exponentially and the qCO₂ increased exponentially in saline soils. They considered that osmotic stress was the main cause of the adverse effects on the microbial biomass. A significant negative correlation between EC and the ratio of C_mic to soil organic C and a significant positive correlation between EC and the qCO₂ were also observed for acidic saline soils (Sardinha et al. 2003). These findings suggested that the increased electrolyte concentration induced by the application of LSS compost was the main cause of the adverse effects on soil microbial biomass. To verify this assumption, the ratio of C_mic/T − C and the qCO₂ were plotted against EC (Fig. 5a,b). The ratio of C_mic/T − C decreased significantly and the qCO₂ increased significantly as EC increased. The EC was strongly correlated with the total amount of water-soluble cations (Fig. 1), and Ca²⁺ was the predominant water-soluble cation (Table 4). The amount of water-soluble Ca²⁺ also showed significant correlations with the ratio of C_mic/T − C and the qCO₂ (Fig. 5c,d). The correlation coefficients (r) were higher for the amount of water-soluble Ca²⁺ than for EC. These results indicate that the electrolyte concentration associated with the amount of water-soluble Ca²⁺ was the major factor affecting the microbial biomass in the soils amended with LSS compost. Osmotic stress was most probably the cause of the adverse effects on microbial biomass in the LSS compost-amended soils in the same way as for the saline soils. An imbalance of mineral nutrients may also inhibit microbial growth in the LSS compost-amended soils.

The ratio of ergosterol/C_mic is considered to represent the proportion of fungal biomass in the total microbial biomass in soil because the amount of ergosterol is closely related to fungal biomass. Khan and Scullion (2000) have found that in soil samples incubated with heavy metal-enriched sewage sludge, the input of heavy metals increased the amount of ergosterol and decreased the ratio of bacterial phospholipid fatty acids (PLFA) to fungal PLFA. These findings suggested that the heavy metals derived from sewage sludge increased the proportion of fungal biomass in the total microbial biomass. In contrast, although significant seasonal fluctuations were observed, an increase in the ratio of ergosterol/C_mic was not confirmed in the present study. Sardinha et al. (2003) reported a negative correlation between EC and the ratio of ergosterol/C_mic for acidic saline soils. A decrease in the proportion of fungal biomass in the total microbial biomass was also found in alkaline saline soils using PLFA analysis (Pankhurst et al. 2001). Additional information that the fungal/bacterial ratio measured using the selective inhibition technique decreased significantly with increasing pH was provided by Bååth and Anderson (2003). Thus, the increased electrolyte concentration along with the high soil pH may inhibit fungal growth in soils that received LSS compost.

CONCLUSIONS

The application of LSS compost resulted not only in a decrease in the ratio of C_mic/T − C but also in an increase in the qCO₂. These results revealed the adverse effects of LSS compost on soil microbial biomass. The analysis of heavy metals indicated that the total concentrations of Cd, Cu and Zn in the soils used in the present study were below the levels known to exert adverse effects on soil microbial biomass. In contrast, EC increased in the LSS compost treatments, and this increase was ascribed to an increase in the amount of water-soluble Ca²⁺. With the increase in EC and in the amount of water-soluble Ca²⁺, the ratio of C_mic/T − C decreased and qCO₂ increased. It was, therefore, suggested that the electrolyte concentration associated with the amount of water-soluble Ca²⁺ was the major factor affecting microbial biomass in soils amended with LSS compost.

ACKNOWLEDGMENTS

We thank Ms J. Tazawa and Ms C. Tarusawa for their technical assistance. B. Zhou thanks the Aomori Prefectural Government for granting him a visiting fellowship during the course of this study.