Plant inputs of carbon to metal-contaminated soil and effects on the soil microbial biomass

Plant inputs of carbon to metal-contaminated soil and effects on the soil microbial biomass

Soil Bid. Biockm. Vol. 23. No. 12. pp. I16‘hl177. hntcd in Great Britain. Au righu reserved 1991 00350717/91 s3.00 + 0.00 Copyright c 1991 Pcrgamon...

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Soil Bid. Biockm. Vol. 23. No. 12. pp. I16‘hl177. hntcd in Great Britain. Au righu reserved


00350717/91 s3.00 + 0.00

Copyright c 1991 Pcrgamon Press pk



K. CHANDER* and P. C. BRwKEst Soil Science Department,


Institute of Arable Crops Research, Rothamsted Harpenden. Herts AL5 UQ. U.K. (Accepted 28 June




Summswy-The amounts of soil microbial biomass in metal-contaminated (high-metal) soils of the Wobum Market Garden Experiment in the U.K. are now about half those in similar uncontaminated (low-metal) soils from the same experiment. The metal-contamination was caused by applications of metal-rich sewage-sludge which ceased about 30 yr ago. Soil metal concentrations in the high-metal soils are now at. or a little above, current European Community limits. This work was designed to see if heavy metals decreased soil microbial biomass by decreasing the input of plant material to the soil or if the synthesis of microbial biomass is less efficient in the presence of heavy metals. Either or both of these mechanisms could explain the effects of heavy metals on microbial biomass in the Woburn experiment. Sunflower (Helianthus annus L., cultivar Sunbred 246) seedlings were grown for 31 days under controlled conditions (I2 h day at ZO’C. I2 h night at 17-C) in a low-metal or a high-metal soil from the Woburn experiment. From days 21 to 31 of growth the plants were supplied with “C-labelled CO: on alternate days. The final dry matter yield (shoots plus roots) of the plants grown on the low-metal soil was about 30% nreater than that of the slants urown on the hiah-metal soil. The distribution of total C and “C-labelled ?I between the various $ant anh soil compartments viz. plant shoots, roots, soil microbial biomass and bulk soil. were measured 2 days after the final “COz labclling. The percentage distribution of “C within shoots, roots, soil microbial biomass and bulk soil was quite similar in both soils. Plant-derived “C-labelled organic C inputs into the high-metal soil were about 20% less than in the low-metal soil. About 35% less of this “C-labellcd C was in the microbial biomass in the high-metal soil than the low-metal soil at harvest. The plants caused an increase in forul biomass C of about 22 and 42 fig C g-’ soil respectively in the high-metal and low-metal soil at harvest, about half of which was “C-labelled in both cases. These increases in biomass were thus in the same ratio as those of the biomass in high-metal and low-metal soils taken directly from the field. These results suggest that both mechanisms (i.e. decreased inputs of C from plants to the soil und decreased etiiciency of conversion of this C into new biomass C) operate in causing smaller biomasses in metal-contaminated soils of the Woburn experiment. The latter mechanism would appear more important than the former.







directly or indirectly. by the heavy metals. These have persisted in the sludged soils, even though the last sludge applications were nearly 30 yr ago. While it is conceivable that the decreased biomass was caused by factors other than metal toxicity this was not considered to be the case. For example, a persistent biocidal organic chemical could have been present in the sludges. This was considered extremely unlikely due to the long period (> 20 yr) between adding the sludges and first observing the decreased biomass. Other evidence that the presence of certain heavy metals in soil affects the amount of biomass is given by Chander and Brookes (199lb). They found that the biomass was decreased by about 40% in both a sandy loam and silty loam soil which had received sludge contaminated singly with Cu 22 yr previously. Soils which had rcccived a Zn-contaminated sludge had a similarly decreased biomass. In contrast the biomass was not decreased in soils which received uncontaminated sludge or Ni-contaminated sludge. Thus, effects on the biomass were due to the presence of different metals in the sludges, not to organic residues, since the same sludge was added to each


and Wedderburn, 1975) which received sewage-sludge contaminated with heavy metals during 1942-1961 were reported to contain, more than 20yr later, only about half the amount of microbial biomass as other soils from the experiment which received farmyard manure or inorganic fertilizer from 1942 to 1967 (Brookes and McGrath. 1984). Since then all soils have received inorganic fcrtiliser only. A variety of crops have been grown throughout the cxperimcnt and soil metal concentrations are now at, or a littlc above. current EC permitted limits (Commission of the European Communities. 1986). Reasons for the smaller microbial biomasses in the metal-contaminated soils were not investigated by Brookes and McGrath (1984), although it was assumed that the decreases in biomass wcrc caused, (Johnston

*Permanent address: Soil Science Department, Agricultural University, Hisar. India. tAuthor for correspondence.






and P. C. Bncotcrs

plot, differing only in metal content. Similarly we consider that the effects we measure here are due to the heavy metals in the soil from Wobum and not to other factors. Two possible mechanisms which could account for the effects of the heavy metals are: (1) that they are directly toxic to the soil microbial biomass. This may decrease the amount of biomass synthesised per unit of substrate or decrease the longevity of the microbial cells. and (2) that the metals may exert direct toxic effects upon the plants themselves, leading, in turn. to decreased inputs of plant-derived substrates (e.g. roots, root exudates) to the soil and consequently to smaller soil microbial biomasses. The possibility of direct toxicity of metals affecting the biomass was investigated by adding equal amounts of “C-labelled glucose or maize to metal-contaminated and non-contaminated soils from the Woburn experiment and measuring biomass over the next 50 days following glucose addition or 100 days after adding maize. Significantly less ‘JC-labelled biomass was formed per unit of added substrate in the metal-contaminated soils and this biomass had a much faster respiration rate than that in similar non-contaminated soils (Chander and Brookcs, 199lb). This suggests that the conversion cfhcicncy of substrates to new microbial cells was lower in the metal-contaminated soil, which may help to explain the finding of smaller biomasscs in the metal-contaminated Woburn soils. Howcvcr, it is also known that heavy-metals (albeit at higher soil conccntrations than in the Woburn cxpcrimcnt) are toxic to plants. causing dccrcascs in plant yields (Cunningham et 111..1975; Wchbcr. 1980). Thus it seems likely that if plant yields arc dccrcascd in mctal-contaminatsd soils, then total plant dcrivcd C inputs to the soils would also be decreased. leading in turn to smaller biomassrs. It is known that the availability of fresh substrates is a major rcgulutor of the growth of soil microbial biomass (e.g. McGill et ul., 1981; Jcnkinson and Ladd, 1981; Mcrckx et of., 1985, 1987; Hula1 and Saucrbcck. 1986; Martens, 1990). Measurcmrnt of the total C input to the soil by a growing plant includes the root C at harvest, plus the C relcascd by roots as exudates, sloughed offor dying root cells and the mineralization of these inputs (Johncn and Sauerbeck, 1977; Whipps and Lynch, 1983; Helal and Sauerbcck. 1986). The use of “C tracer techniques has proved useful in measuring such inputs to soil (Helal and Sauerbeck. 1983; Dinwoodie and Juma. 1988; Martens. 1990). In such studies. intact plants are given “CO, of known specific activity in a growth chamber where the root and shoot regions arc scparatcd by a gas-tight seal, so that CO, respired from the soil can be trapped and measured (c.g. Johncn and Sauerbcck, 1977; Whipps and Lynch, 1983). Many expcrimcnts have been done where “CO1 is supplied to plant shoots, either continuously (Martin, 1975; Whipps and Lynch, 1983; Mcrckx et (II.. 1986a.b. 1987; Martens, 1990) or as a single pulse (Martin and Kemp, 1985; Meharg and Killham. 1990). The choice of method of plant labelling is critical for calculating C budgets. For example, not all parts of pulse-labelled plants become uniformly labelled and consequently the “C entering the soil does not

represent the total inputs of C during the sampling period, as sloughed off or dying root cells will not be uniformly labelled. Thus the “C lost from roots represents only the most recently assimilated plant C. In contrast, in the case of continuously-labelled plants, new photoassimilate and older root material both contribute to the total 14Caccumulating in the soil (Whipps and Lynch, 1983). Continuous labelling therefore permits the absolute plant-derived inputs of C to be determined. A growth-cabinet providing continuous “‘C labelling was not available for this work but a series of pulse labels at regular intervals may provide reasonable estimates of total plant C inputs over the entire labelling period (Meharg and Killham. 1990). In our work the total inputs of plant-derived C to metal-contaminated and non-contaminated soils were measured using 5 pulse-labels, each of I2 h, over a period of IO days. The aim was to see if there were decreased plant-C inputs. and if such decreases could account for the smaller biomasses in the metalcontaminated soil of the Woburn experiment. MATERIALS AND METHODS


Soils wcrc collected ((r-23cm depth) in July 1990 from plot 2, which had received farmyard manure from 1942 to 1967 (low-metal soil) and plot 39, which had rcccivcd metal-contaminated sewage-sludge from 1942 to 1961 (high-metal soil) of the Woburn Market Garden Expcrimcnt. The soils wcrc stored moist at 5°C until use, after removing plant material. stones and obvious soil fauna. They wcrc then sicvcd (< 2 mm), adjusted to 40% of water holding capacity (WHC) and conditioned for 7 days at 25°C over water and soda-lime in air-tight metal bins before USC. Other portions of the soil were air dried and then finely ground before chemical analysis. Soil organic C was measured as described by Kalcmbassa and Jenkinson (1973), total N after Kjeldahl digestion (Bremner. 1965) and soil pH using a 1:2.5 soil: water ratio. Total soil metal contents were measured as described by McGrath and Cunliffc (1985). A brief description of the soil properties is given in Table I. Growth room

The plants were grown, before and during 14Clabelling, in a purpose-built constant environment Table










Experiment Low-metal PH Organic Total Clay

C (%) N (%)




0.16 9.0

Loamy Total


metal (jrg



concentration g-’













7.05 I .b2




7.05 I.25











of carbon and microbial



Fig. I. The grown chamber: I, perspcx box; t. cooling coil: 3, magnetic stirrer; 4, beaker with phosphoric acid; 5, glass funnel with Na,“CO,; 6. glove; 7. flasks with plants; 8, fan; 9, silicone-oil manometer; IO, bcakcrs with silica-gel; 11,inlet tube: 12. outlet tube; 13, thermometer: 14, door with O-rings and screws (not to .scillC).

room. The room was dcsigncd to opcrato at slightly below atntosphcric prcssurc to minimisc the escape of radio~l~tiv~ gas should a leak occur. Light intensity was about 20.0~ ix, and the room had a 12 h day tcmpcraturc of 22 ‘C and I2 h night tempcraturc of 17 c.

The chamber. 46 x 61 x 91 cm. was made from clear perspcx sheet and was that originally designed and used by Jenkinson (1960). For this work it was modified as shown in Fig. I. The growth chamber was set up in the growth room under the above environmental conditions.

“‘CO. was generated within the growth chamber by reacting together aliquots of Na,“CO, solution and I M phosphoric acid. The Na,“CO, was prepared by adding 27.18 MBq iJC (as NarYO,) to 5 1. of 0.5 M Na,CO, stock solution. which was then thoroughly mixed and stored at 5°C. Thus the specific activity of “C in Na,“CO, stock solution was 906. I kBq g-i C. On each day when the ptants were to be labelled, the quantity of Na,“CO, solution required to produce “CO? equal to the maximum amounts that could lx assimilated by the plants during a I2 h photoperiod was calculated. This was done by assuming an average uptake rate of CO? by the plants of 20 /rrnol CO* m-j s-i (Lawlor er at., 1989) and from estimating the total leaf-area of all the plants to be labellcd. The required volume of Na,iJCO, solution was placed in a 500ml separating funnel and the phosphoric acid in a 1 litre beaker with a magnetic stirrer (Fig. I). The Na2“C03 solution was mixed with the

acid dropwisc, with continuous stirring. The speed of Na,“CO, addition to the acid was adjusted by hand via a glove so that it took about IQ-12 h to completely generate the required amounts of “‘CO: from the Nari4C0, solution on each day the plants were labcllcd. A fan was run continuously to mix the “CO3 quickly and uniformly throughout the chamber (Fig. I).

Thirteen flasks of both low- and high-metal soil wcrc prepared by adding a mixture of moist soil, conditioned as above (containing 45Og soil on an oven-dry basis), and 225 g acid washed quartz (S-IO mm) to specially rnodifitd Pyrex glass Buchner flasks. The flasks contained a central tube for supplying water and for flushing air through the soil. Ten flasks of each soil were used for growing plants, the others remained unplanted as controls. Each flask to be planted was fertilized with 1I .25 mg N as NHINO, and II.75 mg P as KH,POo. (equivalent to 50 kg N and P ha-‘) and the soils adjusted to 55% of full WHC. Sunflower seeds (~e/iQ~t~u5 amtrcs, L., cultivar Sunbred 246) were germinated for 2 days in glass Petri dishes, lined with damp filter paper. One seedling was planted in each flask. which were all then covered with aluminium foil, except at the mouth. A second application of II.25 mg N was given to each flask, with irrigation water, 14 days after planting. At 18 days after planting, a rubber bung was inserted into the mouth of each flask. The bung contained a hole through which was inserted the central tube, another central hole for the plant stem and was slit to allow it to be wrapped around the plant stem without damage. The bung-openings were



Fig. 2. Sealed flask containing growing plant: I, Buchner flask: 2. sunflower plant; 3. side-arm for collecting respired CO, and for irrigating soil surface: 4. central tube for flushing soil and head space; 5. rubber bung with openings for plant slcm and central tube; 6. vaseline-parattin seal.

then scaled with a warm mixture of vasclinc and low melting lcmpcraturc paraffin wax in a proportion of 5: I (w/w). The seals wcrc tcstcd for leaks by flushing the flask with air under water. Carbon dioxide cvolvcd from the soil was thcrcforc trapped in the flask and it was rcmovcd as dcscribcd later. Figure 2 illustrutcs the cxpcrimcntal design of a scaled flask containing a growing plant. Sunflower was chosen as the test-plant for two reasons. Firstly, it has a smooth straight stem. which makes scaling the flask easier and, secondly, it grows rapidly so it should provide rcasonablc C inputs to the soils over the total period of 60 h when the plants were supplied with YZ02. Labelliny of planrs wirh “CO,

Twenty-one days after planting, 5 flasks of each soil were randomly selected and then transferred to the growth chamber. All material required for “COz gcncration was also placed in the chamber. The chamber was then closed and CO,-free air was circulated through it via the inlet and outlet tubes (Fig. I). The pressure in the chamber was adjusted slightly below atmospheric pressure, as indicated by a silicone-oil manomctcr (Fig. I). The “CO? was then gcneratcd as dcscribcd above. The plants were given 5 pulses of ‘JCOz, each for I2 h in the light, on altcrnatc days, starting with the 21 day old plants. Thus the plants rcmaincd in a “CO2 or “Cot atmosphere for alternate days ov*er the 21-31 day period. The temperature inside the chamber was kept similar to that in the growth-room by regulating the cold water flow through the cooling coil mounted inside

P. C. Brtoo~~s the chamber (Fig. I). Humidity was not controlled to fine limits. However, much of the water transpired by the plants was condensed on the cooling coil or was absorbed into the silica-gel placed in beakers at several places inside the chamber. The day after each labelling, CO,-free air was again pumped continuously through the chamber for about I h by bubbling the exhaust through a 2 M NaOH trap to remove non-assimilated ‘“CO: or any respired by the plant shoots. The chamber was then opened and the plants removed to the growth room itself. Carbon dioxide produced by soil and root respiration and held in the flask was trapped in 2 M NaOH by flushing each flask for I5 min with C02-free air. During CO, collection at day 24, one flask of the high-metal soil accidentally received NaOH due to back-suction and consequently the growing plant was damaged. This damaged replicate was removed and thus decreased the replicates of the high-metal soil to four for further labelling. A separate NaOH trap was assigned to each flask and used to trap CO1 held in that flask due to soil or root respiration. The NaOH in each trap was unchanged throughout until the plants were finally harvested. 2 days after the last pulse. After each CO2 removal, the flasks were weighed and water corresponding to the amount lost was added half through the side-arm of the flask at the soil surface and half through the central tube, to moisten the base of the soil. AflUlpCS

The plants were harvcstcd after 3 I days of growth. The shoots were cut off, dried at 80’C for 48 h and weighed. Roots and soil were scpardtcd by gcntlc shaking and any small root fragments remaining were carefully removed by hand picking. The roots were washed with water. dried as above and weighed. A sub-sample of soil from each pot was also dried similarly. The dried roots, shoots and sub-samples of the soil wcrc ground separately in a mill and their “C contents dctcrmined by burning in pure OI at 1000°C and trapping the “‘CO1 in IOml Carbasorb (United Technologies, Packard). The trapped “‘CO1 was mixed with 5 ml tolucnc-based scintillation fluid (Pcrmatluor V) in an automatic system (Packard TRI-CARB) and the “CO: in the mixture dctcrmined by liquid scintillation counting. The “CO, evolved from soil and root respiration and trapped in NaOH was analysed by mixing 0.5 ml aliquots of the NaOH solution with 20 ml RIA scintillation cocktail (Fisons plc). The mixtures were stored in the dark for several hours, until chcmilumincscence could not bc detected. Each sample was then counted for 5 min on a scintillation counter. The other portions of the sieved soils were kept moist and stored at 5’C overnight. Biomass C measurcmcnts were then made by fumigationextraction (Vance ef al., 1987) on each planted or unplanted soil. Three rcplicatcs of each soil were fumigated with ethanol-free chloroform for 24 h at 25’C. After fumigant removal, the soils were extracted with 0.5 M K,SO, for 30 min. Three replicates of each unfumigated soil were extracted similarly at the time fumigation commenced and the filtered soil extracts stored at - l5’C until analysis. Organic C in the soil extracts was measured with a Dohrmann DC

Plant inputs of carbon and microbial biomass Table 2. Dry weights*of shootsand rootsof sunflowerafter growth for 31 days?in the low- and high-metal soils Low-metal



Plant Shoots




Shoot:root *Means

ratio of

in the




Distribution of assimilated “C


3.86 0.89





experiments with wheat or maize of about the same age (Martens, 1990).

The plants grown on the low-metal soil fixed significantly more labelled CO2 than those grown on the high-metal soil; II78 and 813 mg C per plant, respectively, excluding that respired by the shoots. which was not measured. Thus about 40% more “C-labelled C was in the shoots of low-metal than high-metal soils. The corresponding differences for the other components were 35% more for roots, 51% more for respired YOz, 39% more for the soil residue and 95% for the microbial biomass of the low-metal than high-metal soil respectively (Table 3). However, the distribution of “C within these different components was quite similar in both soils. In both cases about 75% of the net assimilated “C was in the shoots and the remaining 25% was translocated below ground to the roots and other soil components. An approximately similar distribution of photosynthates was reported for wheat and maize of about the same age (Warembourg and Paul, 1973; Martens, 1990). Large proportions of the “C translocated below ground were respired by soil and root respiration; 47 and 49% in the low- and high-metal soil, rcspcctivcly. Of the total ‘VI translocated below ground, a larger proportion (38-47%) was in the root compartment and a rclativcly smaller proportion (about 12%) was in the soil rcsiduc compartment in both soils. Howcvcr. diGrent proportions of soil residual ‘*C were incorporated into microbial biomass in the two soils. Microbial biomass-“C as a proportion of the soil residue ‘Y (i.e. excluding roots) was about 27% in the low-metal soil and about 19% in the high-metal soil. The distribution pattern of the labelled-C between shoots, roots, rcspircd VO, and soil rcsiduc observed in this study with sunflower grown for 31 days is generally similar to the distribution pattern obscrvcd by Martin (1975, 1977) and Merckx ef al. (1985) for ryegrass, clover and wheat and Helal and Saurbeck (1983) and Martens (1990) for maize of about the same age.


4 replicates

in the


tPlants wcrc grown continuouslyin first 20 days prior

to growth


and a normal




5 replicates




least significant


a normal


then altemativcly



for 5 days in the


(P = 0.05).

80 automatic analyser (Wu et al., 1990). Soil microbial biomass C (Bc) was calculated from: 8, = 2.22 Ec where E, = [(organic C extracted from fumigated soil) minus (organic C extracted from unfumigated soil)] (Vance ef al., 1987; Wu et al., 1990). “C in the K$O, soil extracts was determined by mixing I ml soil extract with 20 ml RIA scintillation cocktail, and counting each sample for 5 min on a scintillation counter. “C-labellcd biomass (biomass 14C) was calculated as above. In the work described in this paper it is assumed that the specific activity of labcllcd C in the diffcrcnt soil-plant components arc constant. Thus “C activities wcrc convcrtcd to weights of labcllcd C using the spccilic activity of Na,“CO,.



The dry matters of shoots. roots and total plants grown on the low-metal soil wcrc about 30% larger than those grown on the high-metal soil (Table 2). However, the shoot:root ratttio of the plants grown on both soils was similar, suggesting that the metals produced a proportional decrease in the growth of both shoots and roots. The plants grown on both soils carried eight leaves at harvest, indicating similar devclopmcnt in either. Thus, dilrerences in plant dry weight were the only signs of the effects of the metals; no visible nutrient dcticicncy or toxicity symptoms were observed. The shoot: root ratios observed in this experiment are comparable with those obtained from other “CO1


3. Dirtrlbution*

of “C

in the plant-soil

system after

growth a per




Total organic “C input into the soil The direct separation of soil respiration into the fraction coming from microbial respiration and that coming from root respiration was not attempted.

of sunflower



for 31 dayst

in the low-

and high-metal

soil, cxprcssed

fhsk basis soil


Low-metal-solI % of t&d

C (ma)

High-metal “C












I II.0










I I.8






3. I

0.57 0.12

Soil, root Soil

Microbial “C


rcwdue$ biomass




in Table



in Table



in Table
































by the plant

in soil at harvest




of roots.



1171 Table

4. Total


of sunflower



organic days+



on Ihe low-


5011 “C-labelled

“C-labelled (roou



soil afkr

and high-metal High-metal


gowth soil LSD:









“C-labelled (roots


not included)


in Table



in Table



in Table


Indeed, such measurements have both conceptual and technical problems. The following approach was adopted. Chander and Brookes (199 I b) added uniformly ‘“C-labelled glucose and maize separately to a high-metal and a low-metal soil from the Woburn Market Garden Experiment in the laboratory. ‘JC-labelled biomass C and “COZ-C evolved from the soil were measured. Such data can be used to estimate the roral amount of plant-C which is needed to produce the measured “C-labelled biomass in the high- and low-metal soil in which sunflower was grown in this work. It can thus be used to estimate the proportion of this labcllcd C which is evolved as ‘JCO& from microbial rcspimtion. Total “CO? evolved from the soil and trapped in the flask was thus separated into respiration by using the “microbial” and “root” values calculated from Appendix I. The calculations. given in full in Appendix I, suggest that 28.2% of the total cvolvcd “COZ-C was from microbial respiration in the low-metal soil and 35.9% in the high-metal soil in the work reported in this paper. Total “C-labcllcd organic C inputs to soil wcrc then calculated separately for the low-metal and high-metal soil by adding togothcr total “C-labcllcd C in the soil with or without including V-labcllcd root C. plus “CO1 respired by microbial respiration (Table 4) as calculated in Appendix I. The results clearly indicate that the input of plant-dcrivcd organic C to soil is considerable, corresponding to about IS-17% of the net assimilated label in both the low- and high-metal soils. This study therefore confirms previous observations that considerable amounts of root derived materials arc lost from actively growing plants, which serve as energy rich substrates IO the microorganisms. However, the C inputs were about 24% larger (including C contained in the roots) or 21% higher (omitting root C) in the low-metal soil than the high-metal soil. This strongly suggests that the heavymetals in the high-metal soil exerted direct phytotoxic etfccts on the plant, leading, in turn, to decreased inputs of plant-derived substrates to soil. This may therefore help to explain the finding of Brookes and McGrath (1984) of smaller biomasses in the high-metal soils of the Woburn Market Garden Experiment. The conversion efficiency (E) of “C-labelled plant C inputs into microbial biomass C was calculated from: E = B”C/soil”C

x 100

where B”C = “C-labelled biomass C at harvest; Soil’JC = total plant inputs of “C-labelled C to soil (excluding root “C) at harvest.

From Tables 3 and 4, the conversion of plantderived “C to microbial biomass “C can therefore be calculated as 9.6/75.5 = (12.7% f 0.3 I %) in the lowmetal soil and 4.8159.3 = (8.2 + 0.33)% in the highmetal soil. These differences are statistically significant (P = 0.05). Thus about 35% less biomass C was synthesised per unit of plant C input in the high-metal soil than low-metal soil. There appear to be few similar experiments reported in the literature to compare with our results, but they do seem to generally concur with those available. From the data of Merckx et al. (1985) it can be calculated that about 3.6 and 14.1% of the total inputs of C into soil by wheat plants grown in a sandy and silty-clay loam soil, respectively, were found as microbial biomass-C 42 days later. Similarly, Martens (1990) grew maize in a continuously “COZlabelled atmosphere and measured about 5% of the labelled-C released to the soil by the plants as microbial biomass-C 46 days later. In contrast, Helal and Sauerbeck (1989) calculated, on the basis of a ratio of 4: I for microbial: root respiration (see Appendix), that a rather larger amount (13%) of total photosynthatc was in the microbial biomass in soil which had grown maize for 21 days. The above biomass measurements were all done using the fumigation-incubation method (FI) of Jcnkinson and Powlson (1976), while the biomass mcasurcments reported in this paper wcrc made by the ncwcr fumigation extraction procedure (FE) (Vance ef a/., 1987; Wu et (II.. 19%)). Biomass measurements by FE are more reliable than those made by FI in soils containing signiticant quantities of lahilc and actively-decomposing substrates such as plant &dues (Ocio and Brookcs. 1990).

The amounts of total and ‘JC-labelled microbial biomass (cxprcsscd as pg C g-’ soil) at days 0 (unplanted) and 31 (planted and unplanted) are shown in Table 5. Plant roots were wry carefully removed before analysis by hand-picking. It is cxtremely unlikely that roots would have caused much error in these measurements. The ratio [(biomass C in low-metal soil)/(biomass C in high-metal soil)] was 1.95 in the unplanted soil at day 0, and was unchanged at day 31 in the unplanted and planted soils, which had grown sunflower plants throughout this period. Biomass C in both the unplanted low-metal and high-metal soils had declined by about 45%. 31 days later. Growth of sunflower plants increased the total biomass C in the low-metal and high-metal soil by about 42 and 22 pg C g-’ soil, respcctivcly, and about half of this in both soils increase in biomass was “C-labelled (Table 5). This indicates that the ratios between the sizes of the new biomass which developed on the plant-derived substrates in the low- and high-metal soils wcrc also similar to the ratios of the biomasses under field conditions. However, this bigger increase of both “C-labellcd and total biomass C in the low-metal soil cannot be solely attributed to the higher inputs of plant-derived C. This is because the biomass in the low-metal soil was more efficient at converting substrate to new biomass than that of

Plant inputs of carbon and microbial biomass


Table 5. Total and “C-laklled soil microbial biomass C in the low- and high-metal soils with and without rrowth of sunflower daau for 31 davst Low-metal soil Soil treatment and time of measurement Unplanted (day 0% Unplanted (day 31). Planted (day 31)*

High-metal soil

Total biomass C fun C P-’ soil) 171.4 164.0 206.3

87.9( 12.91): 84.1(21.10): 105.8 (8.33):


I .95 I .95 1.95

“C&belled biomass C fun c K-’ soil) Planted (dav 311.


10.6 (3.23):


*As in Table 2. tAs in Table 2. :Values in parentheses arc LSDs (P = 0.05). #Mean of three replicates

the high-metal soil (Tables 3 and 4). Thus from these results it seems likely that the smaller biomasses in the high-metal field soils of Woburn Market Garden Experiment are due both to the toxic effects of the metals upon the plants themselves, leading to decreased inputs of plant-derived substrates nnd to the direct toxic effects of metals on the microbial population (Chander and Brookcs, 199lb). It seems highly probable that both mcchanisms operate simullancously. Our results suggcsl that the latter mechanism is more important than the former.

especially taking into account the 14C in the damaged plant, these results suggest that the “C analysis was of acceptable accuracy and precision. Acknowledgem~nrs-K. Chaader thanks the Commoawealth Commission and the British Council for financial support. We also thank D. Burns for help in modifying the chamber, J. Franklin for help with growth-room facilities and M. R. Hart and D. S. Jeakiasoa for useful discussion. In particular we thank D. S. Jeakiasoa for his approach 10 the calculaGoa of soil microbial respiration.


The balance sheet of lJC was calculated on the basis of actual mcasurcd “C in the diffcrcnt plantsoil components of 9 plants, with and without the estimated “C content of I plant damaged by NaOH at day 24 which was not measured (Table 6). Overall, about 91% of the total lJC supplied as Na*“CO, was estimated to be rccovcrcd in the soils and plants, thus about 9% of the total “C supplied was unaccounted for. The “CO, respired by the plants when they were kept outside the growth chamber on alternate days was not measured. This may. at least partially. account for the 9% of “C unaccounted for in this work. Overall, Table 6. Balance sheet of “C in Na*“CO, used to produce “COz to label sunflower plants

“C measured in ditTcrenccomponenls Toul “C in Na,“CO, used to produce “CO: “C recovered in shoots’ “C recovered in roots* “C recovered in roil’ “C respired by sod/roots’ during growth “C unused and/or rcspiredt by shoots during growth Total measured in all components Estimated ‘C in ditkent components of plant damaged by NaOH a( day 24: Total “C recovered (including cstimatcd “C in damaged plant:)


Expressed as % of total

9514.1 6072.2 800. I 249.5

100 63.8 8.4 2.6






8X. I





*Sum of measured values from 9 plants. tThis does not include “C respired by the shoots during the period when plants were grown outside the growth chamber. tFor details see Materials and Methods.

Bremacr J. M. (1965) Total nitrogen. In Lferhocls o/ Soil Anu/y.ris (C. A. Black. Ed.). Vol. 2. pp. I 149-I 178. American Society of Agronomy. Madison. Brookes P. C. and McGrath S. P. (1984) EfTects of metal toxicity on the size of the soil microbial biomass. Journal of Suil Science 35. 341-346. Chaadcr K. and Brookcs P. C. (1991~1) Effects of heavy metals from past applications of sewage sludge on soil microbial biomass and soil orgnaic matter accumulation in a sandy loam and silty loam U.K. soil. Soil Biology & Biochemistry 23, 927-932. Chaader K. and Brookes P. C. (199lb) Microbial biomass dynamics during the decomposiGoa of glucose and maize in metal-contaminated and non-contaminated soils. Soil Biology & Biochemistry 23, 909-915. Commission of the European Communities (1986) Council directive on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture, p. IO. Oflciul Journul of the Europeun Communities LISI. Annex IA. Cunningham T. D., Keeney D. R. and Ryan J. A. (1975) Yield and metal composition of corn and rye grown on sewage sludge amended soil. Juurnul of Enrirunmenful Quality 4, 448-454. Diawoodie G. D. and Juma N. G. (1988) Factors aflectiag the distribution and dynamics of “C in two soils cropped to barley. Plunf und Soil 110, I I l-121. Helal H. M. and Sauerbeck D. R. (1983) Method to study turnover processes in soil layers of ditrereat proximity to roots. Soil Biology & Biochemistry 15. 223-225. Helal H. M. and Sauerbeck D. R. (1986) Erect of plant roots on carbon metabolism of soil microbial biomass. Zeirschri/l fir PJlan:enerniihrung und Bodenkunde 149. 181-188. Helal H. M. and Saucrbeck D. R. (1989) Carbon turnover in the rhizosphere. Zeitschri/r fiir Pflunzenermihrung und Bodenkunde 152. 211-216. Jeakiasoa D. S. (1960) The production of ryegrass labelled with carbon-14. Plum und Soil 13, 279-288.



Jenkinson D. S. and Ladd J. N. (1981) Microbial biomass in soil: measurement and turnover. In Soil Biochemistry (E. A. Paul and J. N. Ladd. Eds). Vol. 5. pp.415-471. Dekker. New York. Jenkinson D. S. and Powlson D. S. (1976) The effects of biocidai treatments on metabolism in soil-V. A method for measuring soil biomass. Soil Biology I Biochemistry 8. 209213. Johnen B. G. and Sauerbeck D. R. (1977) A tracer technique for measuring growth. mass and microbial breakdown of plant roots during vegetation. Ecological Bullefin 25. 366373. Johnston A. E. and Wedderburn R. W. M. (1975) The Woburn Market Garden Experiment. 1942-1969. I. A history of the experiment. details of the treatments and the yields of the crops. Part 2. pp. 79-101. Rorhamsred Experimenrol Station Report for 1974. Kalembasa S. J. and Jenkinson D. S. (1973) A comparative study of titrimetric and gravimettic methods for the determination of organic carbon in soil. Journal of rhe Science q/ Food ond Agriculture 24, 1085-1090. Lawlor 0. W., Gimenez C.. Ward D. A. and Young A. T. (1989) Regulation of photosynthetic carbon metabolism in water-stressed sunflower. In Techniques and Neti Derelupmems in Photos.vnthesis Reseurch (J. Barber and R. Malkin. Eds). NATO ASI series. Vol. 168. pp. 591-595. Plenum Press, New York. McGill W. B.. Hunt H. W.. Woodmansee R. G.. Reuss J. 0. and Paustinn K. H. (1981) Formulation, process controls, parameters and pcrformancc of PtlOENIX: a mode of carbon and nitrogen dynamics in grassland soils. In Simulufion o/ Nirrogen Belwiow u/‘ Soil -Plunk Systems (hl. J. Frisscl and J. A. Van Vccn, Eds). pp. 171~ IYI. Pudoc, Wagenmgen. McGrath S. P. and Cunlilfc C. tt. (19X5) A simplilicd model for the extraction of the metals Fe. Zn, Cu. Ni. Cd, Pb. Cr. Co and Mn from soils and scwagc sludges. Joumul I/’ /he Science uJ Feud und Agriculrure 36. 7Y4 7YX. Martens R. (1990) Contribution of rhizodcposits to the maintenance and growth of soil microbial biomass. Soil Riology & Riochemislry 22, I41 -147. Martin J. K. (1975) “C-lab&d material leached from the rhizosphcre of plants supplied continuously with “CO,. SotI Biology & Biochemisfry 7, 395-3’)‘). Martin J. K. (1977) Factors intluencing the loss of organic carbon from wheat roots. Soil Biulugy & Lfiochemisfry 9. l-7. Martin J. K. and Kemp J. R. (1985) The measurement of C transfers within the rhizosphere of wheat grown in field plots. Soil Biology & Biorhemhrry 18. 103-107. Mehrag A. A. and Killham K. (IY90) Carbon distribution within the plant and rhizosphere in laboratory and field grown Lolium perenne at different stages of development. Soil Biolugy~ & Biochemistry 22. 471-477. Merckx R.. Den Hartog A. and Van Veen J. A. (1985) Turnover of root-derived material and related microbial biomass formation in soils of different texture. Soil Biolqgy & Biochemistry 17, 565-569. Merckx R.. Dijkstra A.. Hartog A. D. and Van Veen J. A. (1987) Production of root derived material and associated microbial growth in soil at different nutrient levels. Biology & Ferfiliry of Suils 5. 126-l 32. Merckx R.. Grinkel J. H. V.. Sinnaeve J. and Cremers (I 986~1) Plant-induced changes in the rhizosphere of maize and wheat. I. Production and turnover of root-derived material in the rhizosphere of maize and wheat. P/urn und Soil 96. 85-93. Merckx R.. Grinkel J. H. V.. Sinnaevc J. and Crcmers (1986b) Plant-induced changes in the rhizospherc of maize and wheat. II. Complexation of cobalt, zinc and manganese in the rhizosphere of maize and wheat. Plunr and Soil %. 95-107.

Ocio J. A. and Brookes P. C. (1990) An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the characterisation of the biomass that develops. Soil Biology & Biochemistry 22. 685-694. Sauerbeck D. R. and Johnen B. G. (1976) Root formation and decomposition during plant growth. In Soil Orgunic Moffer Sfudies, pp. 141-148. Proceedings of a symposium organ&d by the IAEA and FAO, International Atomic Energy Agency, Vienna, 1977. Vance E. D., Brookes P. C. and Jenkinson D. S. (1987) An extraction method for measuring microbial biomass C. Soil Biology & Biochemisfry 19, 703-707. Warembourg F. R. and Paul E. A. (1973) The use of “CO, canopy techniques for measuring carbon transfer through the plant-soil systems. Plum and Soil 38. 331-345. Webbcr J. (1980) Metals in sewage sludge applied to land and their effects on crops, Inorganic Pollution and Agricullure, Reference Book 326. pp. 222-234. Ministry of Agriculture. Fisheries and Food, HMSO, London. Whipps J. M. and Lynch J. M. (1983) Substrate flow and utilization in the rhizosphere of cereals. New Phytologisr 95, 605-623. Wu J., Joergensen R. G., Pommerening B. and Brookes P. C. (1990) Measurement of soil microbial biomass C by fumigationextraction-an automated procedure. Soil Biulogy & Biochemisfry 22, 1167-l 169.

APPENDIX Culculufion

of Microhiul


Buckgrutmd The aim is to calculate the amount of CO:-C respired by the biomass due to metabolism of plant-derived C. Chander and Brookes (199lb) added 10OOr~g C g-’ soil as “Clabellcd glucose and maize separately to a high-metal and a low-metal soil from the Woburn Experiment and measured “CO,-C evolved and biomass “C during the next 50 days (glucose) or IO0 days (maize). Their results for the O-10 day period aficr substrate addition arc used here to represent plant-C inputs over the same period.

Glucose oddifion % of added glucose-“C in biomass at day IO Low-metal High-metal

soil soil

I I.5 8.2 Mui:e uddilion % of added maize-“C in biomass at day IO

Low-metal High-metal

soil soil

Low-metal High-metal

soil soil



% of added glucose-“C evolved as “CO * in O-IO days 52.3 57.8

% of added maize-“C evolved as “CO * in O-IO days

10.3 6.4

39.8 46.6

Mean % of added glucose-“C and maize-“C in biomass at day IO

Mean % of added glucose-“C and maize-“C evolved as “CO1 in O-10 days

10.9 7.3

46.0 52.2

From the data in this paper 21.4 pg biomass “C g-’ soil was synthesised from the input of plant-C (Table 5). If this reprcscnts 10.9% of the total inorganic input of labelled

Plant inputs of carbon plant-C plant-C

(see above). then the total input of labelled organic is: 21410.9

x 100 = 196.3 pg “C g-’

and if the biomass respires 46.0% is equivalent to:


input. this

per flask of soil. respir-

evolved from root respiration = 143.740.6 = 103.1 mg per flask

evolved from microbial

respiration = 40.6 mg per flask.

High -merol soil From this paper’s data 10.6 jrg biomass “C 8’ soil was synthesized from the input of plant-C (Table 5). If this represents 7.3% of the total input of labclled plant-C then the total input of labelled plant-C IS: 10.617.3 x 100 = 145.2 pg “C g-’ and if the biomass respires 52.5% is equivalent to: 145.2 x 52.2/100 or 75.8 x 450/1000


of the plant-C

= 75.8 Irg “C g-’

= 34.1 mg “CO&

evolved from root respiration

= 60.9

mg per flask

evolved from microbial

respiration = 34.1 mg per flask.


and “CO,-C


and “CO,-C

= 40.6 mg “CO&


= 95.0 - 34.1

The total “CO,-C evolved from root and microbial ation was 143.7 mg per flask


. .. “CO,-C .


of the plant-C

196.3 x 46 100 = 90.3 pg “C g-’ or 90.3 x 450/1000

and microbial

input. this


per flask of soil.

The total “CO:-C evolved from root and microbial ation was 95.Omg C per flask


This gives ratios of root: microbial respiration of cu. 3: I in the low-metal soil and 2: I in the high-metal soil. Recently. using a comparable approach (J. Swinnen and J. A. Van Veen, pers. commun.). ratios of about 3:l for root:microbial respiration were obtained for soils growing cereals in the field. There are obviously some large assumptions made in obtaining these results. For example. Chander and Brookes (199lb) added glucose and maize as single large inputs. while the plants would have released C more slowly and in different forms. Also. the laboratory incubations were done at 25-C while the plants were grown at a I2 h day tempcrature of 22‘C and I2 h night temperature of l7‘C. The alternative approach. to assume a ratio of 4:1 for microbial: root respiration (Sauerbeck and Johnen. 1976; Helal and Sauerbeck, 1989) gives values of I I5 mg ‘JCO,-C per flask of soil evolved from microbial respiration in the low-metal soil and 76.0 mg “CO& per flask evolved from microbial respiration in the high-metal soil. This apparently simpler approach does not distinguish between the ditTerent ctlicicncies of substrate conversion to biomass and evolved CO? in low-metal and high-metal soil. as shown by Chandcr and Brookes (I991 b). The two approaches thus give conflicting estimates of the proportions of CO: respired which arc derived from root and microbial respiration. Clearly, more work needs to be done to resolve this problem.