Soil microbial biomass estimates in soils contaminated with metals

Soil Bid. Biochem. Vol. Printed in Great Britain

18, No.

4, pp. 383-388,

0038-0717/86 Pergamon

1986

$3.00 + 0.00

Journals Ltd

SOIL MICROBIAL BIOMASS ESTIMATES IN SOILS CONTAMINATED WITH METALS P. C. BROOKES, CAROLINE E. HEIJNEN,* S. P. MCGRATH and E. D. VANCE Rothamsted

Experimental Station, Harpenden, Herts. AL5 254, U.K.

Summary-The validity of the chloroform fumigation-incubation procedure for measuring soil microbial biomass in field soils contaminated with metals (e.g. Cu, Ni, Zn, Cd) was assessed. The metal contamination was the result of past sewage sludge additions and the contaminated field soils now contain metals at about current maximum U.K. recommended levels. The decomposition of native soil biomass or microbial material added after fumigation was little affected by the presence of metals and it was concluded that fumigation-incubation is a reliable procedure for measuring biomass in soils contaminated with moderate amounts of metals. This conclusion was confirmed by direct microscopy: similar soil biomass estimates were obtained by both methods.

INTRODUCTION About 10’ tonnes of sewage-sludge (wet weight basis) are applied annually to U.K. agricultural soils as organic manure. Sludges from industrial areas in Britain are invariably contaminated with appreciable quantities of potentially toxic metals (e.g. Cu, Zn, Ni,

Cd, Pb and Cr) which accumulate in soils with repeated sludge additions (Department of the Environment, 1981). The soil microbial biomass is the agent of breakdown of the organic materials in the sludges and, in general, increases in response to inputs of decomposable materials such as crop residues or animal manures (Jenkinson and Ladd, 1981). However soils from the Market Garden Experiment at Woburn that had received metal-contaminated sludges (high-metal soils) contained less microbial biomass, as measured by fumigation-incubation (Jenkinson and Powlson, 1976), and had lower microbial activity than similar soils receiving comparable amounts of farmyard manure (low-metal soils) during the same period (Brookes and McGrath, 1984; Brookes et al., 1984). These effects were readily detected even though the last sludge application was more than 20 years ago. The soil metal concentrations in the high-metal soils (Brookes et al., 1984) are close to current maximum recommended levels for UK agricultural soils (Department of the Environment, 1981). The high-metal soils also contained significantly less ATP than the low-metal soils, although the concentrations of ATP in the biomasses of the high- and low-metal soils were similar (Brookes and McGrath, 1984) and comparable to published values (Jenkinson et al., 1979; Jenkinson and Ladd, 1981). The constant ATP concentrations in the biomasses measured in both high- and low-metal soils provide *Present address: Department of Soil Science, Agricultural University, De Dreijen 3, 6703 BC Wageningen, The Netherlands.

reasonable confidence that both methods are applicable to biomass measurements in soils contaminated with maximum recommended amounts of heavymetals. However, fumigation-incubation can give inaccurate estimates of microbial biomass if soils have been air-dried, or have received recent additions of fresh substrate or if soil pH is much below pH 4.5 (Jenkinson and Powlson, 1976). Therefore, in view of the known sensitivity of this technique to certain soil conditions it could be argued that the low biomass contents of the high-metal soils were artifacts of the method, i.e. caused by the presence of the metals and not real. We decided to test this by examining the effects of metals on measurement of soil microbial biomass by fumigation-incubation.

MATERIALS AND METHODS Background

The basis of the fumigation-incubation procedure is that the fumigant, by killing the cells of the indigenous soil microbial biomass, renders them susceptible to decomposition by the recolonizing population that develops during the standard 10 day period of aerobic incubation following fumigant removal. Biomass C is then calculated from Fe/kc, Fc being defined as [(COZ-C evolved from previously fumigated soil in 10 days) - (C02-C evolved from non-fumigated soil in 10 days)], and k, is the proportion of microbial biomass C in soil mineralized to CO& during this period. We use a value for kc of 0.45 at 25°C (Jenkinson and Powlson, 1976; Jenkinson and Ladd, 1981). In this study, metals were added to increase soil EDTA-extractable metal concentrations in both lowand high-metal soils by about the amounts already found in the high-metal soils of the Market Garden Experiment. The effects of these additions on respiration in fumigated and non-fumigated soils, and on fumigation-incubation measurements of soil biomass 383

P. C. BROOKESetal.

384

were then measured. If the added metals significantly reduced the magnitude of the flush of COrC following fumigation, this would result in an apparent reduction in the amount of biomass which, in turn, would account for the smaller biomasses and reduced microbial activity reported in high-metal soils (Brookes and McGrath, 1984; Brookes et al., 1984). The decomposition of a water-soluble cytoplasmic extract of yeast, with or without heavy-metal additions, was also compared in high- and low-metal soil. The aim was to see if the decomposition of this extract, intended to represent the cytoplasmic component of the soil biomass, was influenced by the presence of metals. An apparently low soil biomass would be expected if the metals depressed decomposition of this material. The amounts of biomass in the high- and lowmetal soils were also estimated by direct microscopy (Jenkinson et al., 1976) as an independent estimate of the amount of biomass present.

Cu, 10 pg Ni and 5 pg Cd g-’ soil, or with 2.0 ml of a water-soluble cytoplasmic extract of yeast cells. The yeast cytoplasm extract (1000 pg cytoplasmic C g-’ soil), was added only to the fumigated soils, in two forms; either after suspending the lyophilised extract in water, or in a solution of metals, at the above concentrations. All extracts were exposed to ultrasonic treatment to obtain homogeneous suspensions. After addition of the appropriate solutions to soil, water was added to finally adjust soil moisture to 50% WHC. Each treatment was replicated four times. The yeast cytoplasm extract was that used in the experiments described by Powlson and Jenkinson (1976). It contained 42.3% C and 10.8% N and had been stored frozen (- 15°C) until used here. The amended soils were incubated aerobically for 10 days at 25°C in 1.1 1air-tight bottles, each containing 10 ml water and a beaker with 20 ml 1.OM NaOH. After incubation, CO,-C was determined in the NaOH by autotitration with 1.0 M HCl.

Soils The soils had received either farmyard manure (FYM) at 10.4 tonnes ha-’ a-’ organic matter (OM) on a dry matter basis between 1942-67 or anaerobically digested lagoon-dried sewage sludge applied at 16.4 tonnes ha-’ a-’ OM between 1942-61. Vegetable and arable crops were grown on all the plots until 1972, then the experiment was put down to grass which was ploughed in the autumn of 1982. From 1967, all the plots have received annual uniform additions of inorganic fertilizers. Vegetable crops have again been grown from 1983. Thirty O-23 cm depth soil cores were taken from each plot sampled in February 1985, the moist soils hand picked to remove large pieces of plant material and soil animals, then sieved <2 mm. Then soils from the FYM and sludge plots were mixed separately to provide a “low-metal” and “high-metal” bulked soil. The bulked soils were then incubated under moist aerobic conditions (ZS’C, 40% WHC, 7 days), with a beaker of soda-lime to remove CO,, and subsequently stored in loosely sealed plastic bags at 5°C until required. They were then incubated aerobically at 25°C for 24 h with a beaker of soda-lime, before the fumigation-incubation measurements were made. At this stage, portions of the moist soils were frozen at - 15°C to provide samples for total biomass estimates by direct microscopy. Soil pH was measured with a glass electrode using a 1:2 soil to water ratio. Total N in soils was measured by Kjeldahl digestion (Bremner, 1965) and organic C by dichromate digestion (Kalembasa and Jenkinson, 1973). The bulked low-metal soil contained 1.67% C, 0.150% N and had a pH of 7.0. The high-metal soil contained 1.95% C, 0.173% N and had a pH of 6.8.

Microscopic measurement of biomass

Fumigation-incubation

measurements

Portions of the moist high- and low-metal soils (50 g oven-dry) were incubated, with or without alcohol-free chloroform, for 24 h at 25°C (Jenkinson and Powlson, 1976). After fumigant removal, all soils were adjusted to 50% WHC either with water (2.3 ml) or with 2.0ml of a mixture of ZnSO,, CuSO, , NiSO, and CdCl, to give 160 p g Zn, 100 p g

Direct microscopic estimates of biomass in the bulked low-metal and high-metal soil were made using the Jones-Mollison agar film technique with phenolic aniline blue staining (Jenkinson et al., 1976). The frozen bulked soils were thawed immediately before use. Different soil weights were used in anticipation of the different abundances of biomass in the two soils (Jenkinson et al., 1976). Thus four replicate subsamples of the low-metal soil (5.0 g fresh weight, 86.9% dry matter) and the high-metal soil (10.0 g fresh weight, 85.8% dry matter) were each dispersed in 60 ml agar to prepare the agar-films. The conversion factors used to convert direct counts to biomass C were: specific gravity 1.1, dry matter 20% and 46% C. Soil metal analysis

Metals extracted by 50 mM EDTA (Ministry of Agriculture, Fisheries and Food, 1981) and 0.1 M CaCl, (Sauerbeck and Styperek, 1985) were determined in air-dried portions of the amended and non-amended soils after the 10 day incubation using a 1: 5 soil : solution ratio. Extractable metal concentrations in Tables 1 and 2 were obtained by averaging quantities extracted from fumigated and nonfumigated soils after incubation. The extracts were analysed for metals using an inductively-coupled plasma emission spectrometer (ARL 34000). RESULTS

AND DISCUSSION

Extractable soil-metal concentrations

The amounts of metals (Zn, Cu, Ni, Cd) added to the soils at the start of the incubations were chosen to increase the EDTA-extractable metals in both the low- and high-metal soil by about as much as was initially present in the high-metal soil. In fact, the final concentrations of EDTA-extractable metals in both soils after addition and 10 days incubation (Table 1) were rather larger than might be expected from previous field data on these soils (McGrath, 1984). This suggests that the extractabilities of the metals recently added as inorganic salts were greater than the extractabilities of the metals which had been

385

Microbial biomass in soils contaminated with metals Table 1. Recovery by 5Om~ EDTA of Cu, Zn, Ni and Cd added to low-metal and high-metal soils

Metal additions Soil Low-metal

High-metal

Metal

Soil metal concentration With metal Without metal additions additions (fig g-’ soil)

Recovery of added metal (%)

Zn Cu Ni Cd

160 100 10 5

44.05 14.76 2.04 1.13

178.03 100.31 10.08 5.79

83.7 85.6 80.0 93.2

Zn Cu

160 100

139.23 56.54

278.80 143.67

87.2 87.1

z:

10 5

4.69 6.67

15.30 9.42

94.6 86.3

LSD (P = 0.05): Zn = 16.97, Cu = 11.37, Ni = 1.25, Cd = 0.62 *The metals were added to the soils before they were incubated aerobically for 10 days.

added in sewage-sludge many years ago. These metal additions therefore represent a situation of greater potential biotoxicity than the original sludge-treated soils and thus provide a more extreme test of the validity of the fumigation-incubation technique than the original field soils. Recoveries of the added metals were quite similar between metals and between soils, ranging from about 84% for Zn in the low-metal soil to 95% for Cd in the high-metal soil. EDTA was thus very effective in extracting the metals added, although this may not accurately reflect their biological availability. Table 2 shows the amounts of metals extracted from the low- and high-metal soil by 0.1 M CaCl,. Sauerbeck and Styperek (1985) suggested that this reagent provided a better indication of the biological availability of metals in soils than stronger reagents such as EDTA. Recoveries of added metals by CaCl, were much lower than with EDTA and also differed more between metals. For example, less than 1% of the added Cu was extracted from either soil by CaCI,, compared with more than 80% extracted by EDTA. If biological availability is related to CaCl, extractability then it should decrease in the order Cd > Ni > Zn > Cu, irrespective of whether the soil was initially low or high-metal in this experiment. Eflects of added metals on CO,-C evolution

The amounts of CO,-C evolved from nonfumigated low- and high-metal soils in 10 days, without added metals (Table 3), were very similar (62

and 65pgCg-’ soil) despite the much greater amounts of EDTA and CaCl,-extractable metals in the high-metal soil (Tables 1 and 2). However, the microbial populations in both the low- and highmetal soil were equally affected in terms of respiration by an identical addition of Cu, Ni, Zn and Cd, which resulted in a decrease in CO*-C evolution of about 30% in both soils (Table 3). In contrast to the similar rates of respiration in non-fumigated low- and high-metal soils (non-metal amended) (Table 3), fumigation increased COz-C evolution from the low-metal soil to 139pgC gg’ soil, an increase much larger than from the highmetal soil (96 pg C g-’ soil). Assuming a kc of 0.45 (Jenkinson and Ladd, 1981), the corresponding biomass C contents of the non-amended low-metal and high-metal soils were 171 and 68 pg C g-i soil. These results are consistent with those of Brookes and McGrath (1984), who also reported decreased biomasses and ATP concentrations in high- compared to low-metal soils taken from the same field experiment. Adding extra amounts of metals to both low- and high-metal soils before fumigation-incubation also reduced respiration when the soil was fumigated, although the decrease was small (Table 3). Because CO& evolution from the non-fumigated soil was reduced to a greater extent after metal addition, the calculated soil biomass contents of both low- and high-metal soils were both slightly higher following metal addition, than in soils without addition of metal solutions.

Table 2. Recovery by 0.1 M CaCI, of Cu. Zn, Ni and Cd added to low-metal and high-metal soils

Metal addition’ Soil Low-metal

High-metal

Metal

Soil metal concentration With metal Without metal additions additions (pg g-’ soil)

Recovery of added metal (%)

Zn Cu Ni Cd

160 100 IO 5

I .54 0.16 0.12 0.26

37.60 0.74 3.61 3.14

22.5 0.6 34.9 57.6

Zn cu Ni Cd

160 100 10 5

8.44 0.35 1.01 I .47

50.99

26.6 0.7 48.9 61.8

I .oo 5.90 4.56

LSD (P = 0.05): Zn = 3.46, Cu = 0.06, Ni = 0.20, Cd = 0.12 “The metals were added to the soils before they were incubated aerobically for 10 days.

P. C. BR~~KESet al.

386

Table 3. Effect of adding metals to soils on CO,-C evolution from fumigated and non-fumigated on the calculated microbial biomass C content CO& evolved during 10 days after fumigation Metal@ added

Soil

Low-metal +

High-metal

+

Fumigation 62 139 43 129

0

65 96 44 80

LSD (P = 0.05)

Biomass C

(kg C gg’ soil)

0 CHCI, 0 CHCl, CHCI, 0 CHCl,

Increase in CO& evolution due to fumigation (F,)

soil and

77

171

86

191

31

68

36

80

8.2

n160~g Zn, 1OOpg Co, 1Opg Ni and 5pg Cd added g-l soil.

Eflects of heavy-metals in soil on the decomposition of yeast cytoplasm extract

Any factor (e.g. metals) which reduced the ability of the recolonizers to mineralize the biomass in a previously fumigated soil would result in a diminished flush of CO,-C (Fc). Biomass C, calculated from F,, using the conventional k, value of 0.45, would then be erroneously low. Inhibition of the recolonizers by the metals could be exacerbated if the killed population accumulated metals from the soil against a concentration gradient (Doelman, 1986). These possibilities were tested by adding a lyophilized water-soluble cytoplasmic extract of yeast to the lowand high-metal soil after fumigation. It was intended that the yeast cytoplasmic extract should represent the decomposition properties of the cytoplasmic component of the soil microbial biomass, as Powlson and Jenkinson (1976) and McGill et al. (1981) suggested that it is mainly this fraction of the soil biomass that is mineralized during fumigation-incubation. Stainable microbial cell-walls are still apparent more than 50 days after aerobic incubation of a previously fumigated soil (Jenkinson et al., 1976). The results in Table 4 show that the yeast cytoplasmic extract decomposed to the same extent (i.e. about 56% of added yeast-C was evolved as CO,-C in 10 days) in both low- and high-metal soils and, further, that its decomposition was unaffected if metals were added to the yeast cytoplasmic extract before ultrasonification. Assuming that the behaviour of the yeast cytoplasmic extract is representative of

the material from the native soil microbial biomass made decomposable by fumigation, then it follows that k, (i.e. the fraction of soil microbial biomass C decomposed during fumigation-incubation) is not influenced by metals, whether freshly added or already present. These results also indicate that the reason for the slight decrease in CO*-C evolution from fumigated low- and high-metal soils following fresh metal addition (Table 3) was not reduced decomposition of microbial biomass during fumigation-incubation. It is likely that mineralization of non-biomass soil organic matter in both non-fumigated and fumigated soils also decreased following metal-addition (Table 3). This was probably the main factor reducing COr-C evolution from the fumigated soils following addition of the metal solutions. These effects were small however, even though, after addition of fresh metals, soil metal concentrations were in excess of current recommended U.K. levels in the high-metal soil (Department of the Environment, 1981). These results therefore suggest that biomass measurements by fumigation-incubation can be made with reasonable confidence in soils contaminated with metals at, or above, current U.K. recommended levels. Microscopic estimates of microbial biomass in lowmetal and high-metal soils

The best test of the validity of indirect measurements of soil biomass (e.g. fumigation or soil ATP) would be to compare them with results of chemical

Table 4. Decomposition of yeast cytoplasm extract, added with or without metals, in lLmiaafed soil during a 10 day aerobic incubation Decomposition Soil Low-metal

High-metal

Addition

CO,-C evolved @g Cb,-C g-’ soil)

None Yeast” Metals” Yeast + metal

135 678 112 680

None Yeast Metals Yeast + metal

19 635 72 638

LSD (P = 0.05) ‘1000~1g yeast C added g-’ soil. b160 pg Zn, 100 fig Cu, 1Opg Ni and 5 pg Cd

14.6 added gg’ soil.

of added Yeast

(%). 53 56 55 56

Microbial biomass in soils contaminated

analysis of micro-organisms quantitatively extracted from soil. To date, however, it is not possible to extract soil micro-organisms in sufficient amounts, or sufficiently free from contamination, for accurate analysis. The best that we could do was to compare the amounts of biomass estimated by current indirect methods with those estimated by direct microscopic examination of appropriately stained soil films. To make such comparisons, biovolumes, measured microscopically, are converted to biomass C, assuming values for the specific gravity, cellular C content and water content of cells. However, the principal problem in estimating microbial biomass C by direct microscopy is distinguishing living organisms from dead organisms, or other organic material, from their responses to stains. Subjective decisions as to whether the objects viewed under the microscope were living organisms when the films were prepared have to be made, with more or less confidence (Jenkinson and Ladd, 1981). However, despite these problems, direct microscopy is a valuable technique, being the only direct check at present of the validity of indirect biomass measurements in soil. Jenkinson et al. (1976) found good agreement between biomass measured by direct microscopy (direct counts) and by fumigation-incubation for six arable, two grassland and one woodland soil. Similarly, Nannipieri (quoted in Paul and van Veen, 1978 and Jenkinson and Ladd, 1981) and Tesaiova and Repova (1984) found reasonable agreement between soil microbial biomass measured by these two techniques. In contrast, Schniirer et al. (1985) found biomass measured by fumigation-incubation to be only half, or less, that measured by direct counts. However, their definition of biomass was considerably more liberal than that of Jenkinson et al. (1976), who excluded brown hyphae not stained with phenolic aniline blue: Schniirer et al. included such brown hyphae in their measurements. Table 5 shows that biomass estimates by direct counts were 23% higher than with fumigationincubation in the low-metal soil; in the high-metal soil the difference was greater, biomass by direct counting being 79% greater. Two reasons can be offered for the larger biomasses measured by direct counts compared to fumigation-incubation in this study. Firstly, necromass (dead biomass) may have been counted; secondly, the greater discrepancy in the high-metal soil may be because a higher soil-tosolution ratio was used in preparing the soil film from this soil. This was done to obtain a sufficient density of organisms for statistically valid counts in the high-metal soil. The larger amount of mineral mate-

with metals

387

360

300 [

a

s

Trn 250-

“E *E 200 10 ‘0

1 a .g i 3

low-metal soil

150-

loo-

so-

0.01

0.1

1.0 Volume

of organisms

10

100

(ym3)

Fig. 1. The relationship between cumulative biovolume (y) and organism volume (x) for spherical organisms in low-metal (-M) and high-metal (+M) soils. The regression equations were (-M); y = 85.17 log,6 + 143.8 (94.7% of variance accounted for) and (+M); y = 52.47 log,,+ + 82.06 (94.1% of variance accounted for).

rial present in the agar film may have reduced the accuracy of direct counts in this high-metal soil, as indicated by the higher standard error for direct counts (Table 5). Despite these discrepancies both methods showed that biomass C in the high-metal soil was about half that in the low-metal soil. This result therefore adds support to the other data presented in this paper indicating that moderate amounts of metals in soil do not greatly affect fumigation-incubation measurements. It also confirms previous results with these soils which showed that biomass measured by fumigation-incubation, in high-metal soils of the Woburn field experiment, was about half that present in the low-metal soils (Brookes and McGrath, 1984). Table 5 also shows the quantities of spherical and cylindrical biomass in the low- and high-metal soils. The (fungal biomass C)-to-(bacterial biomass C) ratio is also shown, bacterial biomass being defined as that of spherical organisms with diameter less than 1 pm. There was little evidence that the fungal-tobacterial ratio was influenced by metals in these soils. Jenkinson et al. (1976) showed, for spherical organisms in eight soils, that if the soil biovolume was divided into equal volume classes on a logarithmic basis, then each class contained the same biovolume. This relationship occurred despite the large difference in total numbers of organisms in different classes; for example, organisms in the 0.1-l pm3 class were 100

Table 5. Microbial biomass C @g C g-’ soil) in low-metal and high-metal soil, measured by direct microscopy and CHCl,-fumigation Total biomass C CHCI,-fumigation

Biomass C

Direct microscopy

Soil

Spherical biomass C

Cylindrical biomass C

Bacterial biomass C’ Fungalb/bacterial biomass C ratio

(fig C g-’ soil)

Low-metal

171

High-metal

68

21 I

+_5.8

122 + I5

“All spheres up to l.Opm diameter assumed to be bacteria. bAll cylindrical organisms assumed to be fungi.

145 +_8.1 93 + 14.0

65 + 8.1

IO * 0.2

6.4

30+ 1.7

6 + 0.3

5.4

P. C. BR~OKESet al.

388

times more numerous than organisms in the 10-100 pm3 class. We found the same relationship for both high- and low-metal soils, which differed only in slope, due to the lower total biomass in the highmetal soil (Fig. 1). The regressions for the low- and high-metal soil both accounted for about 95% of the variance of the data. Jenkinson el al. (1976) were unable to satisfactorily explain the reasons for the close relationship between biovolume and logarithmic size class and, similarly, we can offer no satisfactory explanation. Acknowledpements-We thank P. W. Hellon and N. D. Sills for help kith the experiments and D. S. Jenkinson (Rothamsted), E. T. Elliot and D. A. Klein (Colorado State University, U.S.A.) for helpful discussion.

for measuring soil biomass. Soil Biology & Biochemistry 8, 209-213.

Jenkinson D. S., Davidson S. A. and Powlson D. S. (1979) Adenosine triphosphate and microbial biomass in soil. Soil Biology & Biochemistry 11, 521-527.

Jenkinson D. S., Powlson D. S. and Wedderburn R. W. M. (1976) The effects of biocidal treatments on metabolism in soil. III. The relationship between soil biovolume, measured by optical microscopy, and the flush of decomposition caused by fumigation. Soil Biology & Biochemistry 8, 189-202. Kalembasa S. J. and Jenkinson D. S. (1973) A comparative study of titrimetric and gravimetric methods for the determination of organic carbon in soil. Journal of the Science of Food and Agriculture 24, 1085-1090. McGrath S. P. (1984) Metal concentrations in sludges and soil from a long-term field trial. Journal of Agricultural Science (Cambridge)

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