Copper and zinc fractions affecting microorganisms in long-term sludge-amended soils

Abstract

The influences of Zn and Cu on soil enzyme activities (acid phosphatase, alkaline phosphatase, arylsulfatase, cellulase, dehydrogenase, protease (z-FLase), urease, β-D-glucosidase and β-D-fructofuranosidase (invertase)) and microbial biomass carbon were investigated in agricultural soils amended with municipal sewage sludge or compost since 1978. The trace metals in the soils were fractionated using a sequential extraction method. Long-term application of the sewage sludge and composts caused accumulations of Cu and Zn in the soils, ranging from 140 to 144 and from 216 to 292 $\text{mg}\text{kg}{}^{\mathrm{-1}}$, respectively. The percentage of Cu was highest in the NaOH- and HNO₃-extractable fractions (44–51% and 38–46%, respectively), while the percentage of Zn was highest in the HNO₃- and EDTA-extractable fractions (65–83% and 11–32%, respectively). Although the percentage of the bioavailable fractions (sum of KNO₃+H₂O-, NaOH-, and EDTA-extractable amounts) of Cu (53–64%) was higher than that of Zn (15–37%), the percentage of the most labile fractions (KNO₃+H₂O) of Zn (2.1–5.9%) was larger than that of Cu (1.1–2.4%). The size of the microbial biomass carbon increased with the application of sewage sludge or compost. For some enzymes, however, the ratio of the enzyme activity to microbial biomass was lower in the soils amended with sewage sludge or compost than that in the control soil. The soil enzyme activities were more adversely affected by Zn than by Cu. From a multiple regression analysis, it was found that dehydrogenase, urease, and β-D-glucosidase activities were reduced by the KNO₃+H₂O-extractable fraction of Zn in the soils. These microbial activities seem to be sensitive to Zn stress, indicating the possibility that they might be useful bioindicators for evaluation of the toxic effects of Zn on microorganisms in the soils.

Introduction

Huge amounts of sewage sludge have been produced, especially in advanced countries. The sewage sludge contains appreciable amounts of plant nutrients (especially P and N) and agronomically beneficial organic matter. The application of sewage sludge to agricultural land enables us to reduce the use of artificial fertilizer and to make good use of resources. Actually, the application of sewage sludge on agricultural land has been practiced in many countries (McGrath et al., 1994). The sewage sludge may, however, contain potentially harmful trace metals. Hence, repeated application of the sludge can lead to the accumulation of trace metals in soils, causing adverse effects on soil microbial activity and crop productivity (Baath, 1989, McGrath et al., 1994, McGrath et al., 1995, McBride, 1998, Giller et al., 1998). It is generally accepted that accumulated trace metals reduce the amount of soil microbial biomass (Brookes and McGrath, 1984, Chander et al., 1995) and various enzyme activities (Mathur and Sanderson, 1980, Kandeler et al., 1996), leading to a decrease in the functional diversity in the soil ecosystem (Kandeler et al., 1996). However, the observed effects are not consistent among sewage sludge-treated soils. Fliessbach et al. (1994) suggested that low-metal sludge applications increased the microbial biomass carbon (C_mic) and the soil microbial activity. However, Dahlin et al. (1997) showed that the influence of the trace metals on the soil microbial properties was statistically significant even in the low-metal-containing sewage sludge-treated soils. Baath et al. (1998) showed that the pattern of phospholipid fatty acid was significantly altered by high levels of Cu, Ni, and Zn in the sewage sludge-treated soils with no effects on the metabolic capacities of the microbial community. Chander and Brookes (1991b) reported that C_mic decreased for the low-Cu treatment, while no effects were found for the high-Ni and high-Zn treatments in soils amended with sewage sludges enriched with single metals.

In field studies, it is necessary to distinguish the influence of trace metals from that of other soil properties because many factors other than trace metals also affect the soil microorganisms (Palmborg and Nordgren, 1996, Palmborg et al., 1998). Various soil enzyme activities have been measured to examine the influence of trace metals on the microorganisms in soils. The soil enzymes consist of intracellular and extracellular enzymes (Burns, 1982, Dilly and Nannipieri, 1998). The extracellular enzymes can be stabilized in a three-dimensional network of organo-mineral complexes (McLaren, 1975, Burns, 1982) and maintain their activities (Sarkar et al., 1989, Lahdesmaki and Piispanen, 1992, Dilly and Nannipieri, 1998). The activities of these extracellular soil enzymes are affected by the soil constituents (Gianfreda and Bollag, 1994), pH (Quiquampoix, 1987, Eivazi and Tabatabai, 1990), and trace metals (Eivazi and Tabatabai, 1990, Geiger et al., 1998b) because these factors modify the conformation of the enzymes (Staunton and Quiquampoix, 1994, Leprince and Quiquampoix, 1996, Geiger et al., 1998a). Furthermore, Eivazi and Tabatabai (1990) assumed that metal ions might inhibit enzyme reactions: (i) by complexing the substrate, (ii) by combining with the protein active group of the enzymes or (iii) by reacting with the enzyme–substrate complex. It is also known that pH affects enzyme stability in soils (Frankenberger and Johanson, 1982). Therefore, we must also consider soil properties along with the metals in order to examine the influences of the metals on the soil enzyme activities.

Chander and Brookes, 1991b, Fliessbach et al., 1994 considered that the ratio of microbial biomass carbon (C_mic) to total soil organic carbon (Org-C) was a useful indicator for evaluating the influence of trace metals on the microorganisms in soils amended with sewage sludge. The C_mic/Org-C ratio is occasionally used to eliminate the effects of organic matter content of the microbial biomass size and to reflect the influence of only the metal-dependent part of the microbial biomass. Similarly, the ratio of enzyme activity to microbial biomass can be used as a sensitive index for evaluating environmental stress (Tscherko and Kandeler, 1997, Aoyama and Nagumo, 1996, Aoyama and Nagumo, 1997). The combinations of microbial biomass with soil organic matter and soil enzyme activities with microbial biomass might provide a more sensitive indication of trace metal contamination than either enzyme activity or microbial biomass measurements alone.

Trace metals in soils are present in various forms due to interactions with various soil components; therefore, the total concentrations in soils cannot provide a precise index for evaluating their influence on soil microorganisms (Welp and Brummer, 1997; Kunito et al., 1997a, Kunito et al., 1997b, Kunito et al., 1999a, Kunito et al., 1999b). To date, few attempts have been carried out to reveal the relationships between microbial properties and trace metal forms in soils (Kunito et al., 1998), though many studies have been conducted for crops (see a review of Ross (1994)). Also, it must be noted that many kinds of trace metals in sludge simultaneously affect the microorganisms in the soils amended with sewage sludge. However, very little work is currently available in the published literature on the subject.

In the present study, soil samples were collected from surface horizons in experimental plots; Cu and Zn had accumulated in these soils (Chino et al., 1992, Goto et al., 1997) due to the application of municipal sewage sludge or compost since 1978. A sequential extraction technique was used to analyze the chemical forms of the trace metals in the soils. Several soil enzyme activities contribute to the biogeochemical cycle of the important elements (C, N, P, and S) (Kiss et al., 1975, Dick and Tabatabai, 1993), whereas the microbial biomass in the soil is a labile reservoir for these elements (Jenkinson and Ladd, 1981). Hence, these parameters were measured to assess the influence of the forms of the trace metals on the microbial-mediated soil processes.