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Microbial biomass dynamics during the decomposition of glucose and maize in metal-contaminated and non-contaminated soils
Soil Bud
Budtern
Vol. 13. No
0038-0717.91 $3.00+
IO. pp. 917-925. 1991
0.00
Copyright C 1991 Pergamon Press plc
Pnntcd m Great Bntam. All nghts reserved
MICROBIAL BIOMASS DYNAMICS DURING THE DECOMPOSITION OF GLUCOSE AND MAIZE IN METAL-CONTAMINATED AND NON-CONTAMINATED SOILS K. CHASDER* Soil Science Department.
AFRC
and P. C. BRooKEst
Institute of Arable Crops Research. Rothamsted Harpenden. Herts AL5 2JQ. U.K.
Experimental
Station.
(Accepted 5 May 1991) Summary-Metal-contaminated soils (produced by past long-term applications of contaminated sewagesludge) from the Woburn Market Garden Field Experiment were previously shown to contain only about half the amounts of microbial biomass as other soils from the experiment which received farmyard manure during the same period. In some cases. the amounts of biomass in the metal-contaminated soils were even smaller than in other soils from the experiment which received inorganic fertilizer throughout. It is possible that the metals were causing decreased efficiency of substrate utilization by the microbial biomass. leading. in turn. to a smaller microbial population. This was investigated in a laboratory glucose and “C-labelled experiment by adding “C-labelled maize shoots (maize) separately to a metal-contaminated and a non-contaminated soil from the field experiment. Microbial biomass C. ninhydrin-N. soil ATP content and CO? evolution were measured during the next 50 days following glucose addition and 100 days following maize addition in both soils. The biomass formed following addition of glucose or maize was consistently smaller in the metal-contaminated soil throughout the incubations. Overall. about I5 -32% less glucose-derived and 25-60X less maize-derived biomass was formed in the mct;ll-contamin;ttcd soil. In contrast, more CO& was cvolvcd from the metal-contaminated soil than from the non-cont;lmin;~tcd soil. This suggests that the biomass in the metal-contaminated soil was less cllicicnt in the uttli~;ttion of substrates for biomass synthesis. It is suggested that this may be a major reason for the smaller biomass in the mct&contaminatcd Woburn soils.
IN’l’WOI)tiC-TION
when micro-organisms cxpericnccd such cnvironmcntal strcsscs. including heavy metals. Large concentrations of heavy metals arc harmful to the growth, morphology and metabolism of microorganisms in oifro (e.g. Babich and Stotzky, 1977, 1982; Bhattachcrjee, 1986; Duxbury. 1981; Rachlin rr 01.. 1982; Siegel er 01.. 1986). The binding of metals onto sites on the cell wall may lead to changes in the morphology of the ccl1 surface, and disrupt normal functioning (Collins and Stotzky, 1989). Heavy metals may also denature proteins (e.g. Gadd and Griffiths, 1978) or destroy the integrity of cell mcmbranes, allowing mctabolites to leak out (Rich and Horsfall. 1963). Organisms living in metal-contaminated environments may therefore need to expend more energy to survive than if they lived in less stressful situations. Thus it might be expected that the cfficicncy of conversion of fresh substrates into new soil microbial biomass may be less cfhcicnt, respiration may be faster and the rate of microbial turnover increased at clcvatcd soil metal contents. If these factors opcratcd, they could help to explain the smaller biomasscs that have been observed in metal-contaminated soils. We therefore have dctcrmincd if heavy metals exert such direct toxic effects on the microbial biomass, using soils from the Woburn Expcrimcnt. This was done by adding “C-labelled glucose and “C-labellcd maize shoots to uncontaminated and metal-contaminated soils and measuring the synthesis of biomass
In 1984, soils from the Woburn Market Garden Expcrimcnt, which last received metal-contaminated scwagc-sludge 23 yr previously (Johnston and Wcddcrburn. 1975) were reported to contain only about half the amounts of microbial biomass found in other soils from the cxpcrimcnt which rcccivcd farmyard manure during the same period (Brookcs and McGwth. 1984). In some cases the biomass in the soils contaminated with heavy metals (e.g. Cu. Ni, Cd and Zn) was even smaller than in soils that had received inorganic fcrtilizcr throughout the experimcnt. The mechanisms producing the smaller biomasses in the metal-contaminated soils [now containing Cu. Ni and Zn at about the EC limit (Commission of the European Communities. 1986). and Cd at about two to three times above it] were not then investigated. However, it is known that cnvironmcntal stresses (such as extrcmcs of pH. tcmpcraturc, salinity or metal content) perturb the division of cncrgy bttwecn growth and maintcnancc in micro-organisms. For example. Killham (1985) and Killham and Fircstonc (1984) reported increased diversion of carbon from biosynthesis to maintcnancc cncrgy rcquircmcnts -___I_
-__-
*Permanent address: Dcpartmcnt of Soil Science Haryana Agricultural University. Hisar. India. tAuthor for correspondence. 917
K. CHANDER and P. C. B~oorcas
918
and CO,-C evolved as the substrates decomposed under laboratory conditions. We used biomass C and biomass ninhydrin-N measurements (by fumigationextraction) and biomass ATP measurements because these methods have been shown (Ocio and Brookes. 1990) to provide reliable biomass estimates in unamended soils and in soils containing recent inputs of readily-decomposable substrates.
MATERIALS
AND METHODS
Soils Soils were sampled to plough depth (0-23cm) in October 1988 from plot No. 2. which had received farmyard manure from 1942 to 1967 (low-metal soil) and plot No. 39, which had received metal-contaminated sewage-sludge from 1942 to 1961 (high-metal soil) of the Woburn Market Garden Experiment. Since then, both soils have received inorganic fertilizer only. The soils were stored moist at S’C until use, after removing plant material, stones and visible soil fauna. They were then sieved (< 2 mm). adjusted to 40% of water holding capacity (WHC) and were equilibrated for 7 days at 25 C over water and soda lime in air-tight metal bins before use. Other portions of soils were air-dried and then finely ground in a Tcma mill bcforc chemical analysis. A brief description of the soil propcrtics is given in Table I.
Soil pH was mcasurcd with a glass clcctrodc using a I : 2.5 soil-to-water ratio. Total soil N was mcasurcd by Kjcldahl dig&on (Brcmncr. IY65) and organic C in plant material and soils by dichromatc digestion (Kalcmbasa and Jcnkinson, lY73). Total metals wcrc dctcrmincd after digestion of the soils with 4: I (v/v) HCI: HNO, (McGrath and Cunlilfc, 19x5). Soil treatments Two separate laboratory experiments were done, one using “C-labcllcd glucose and the other using uniformly “C-labclIcd maize shoots (maize) as substratc. The maize was oven-dried at 35°C for 2 days and then lincly ground (< 160 pm) before use. Three sets of moist low- and high-metal soil (each containing 50g soil on an oven-dry basis) were weighed into 100 ml jars. The first set was used to measure soil microbial biomass C and biomass ninhydrin-N. the second to measure evolved CO, and the third for ATP measurements.
Table
I. Analysis of soils from the Wohurn Erpcnment
PII Organic C (%) Total N (%) Clay (%I TCXllKC Tuotul metal (pg * Zll
CU Ni Cd Cr Pb
Market
Garden
Low-metal 5011
High-mctal soil
7.06
7.06 I .6? 0.16 9.0 Loamy-sand
I.25 0.13 90 Loamy-tand
concmrrorion
’ soil)
107 26 16 I.4 4n 42
419 12s M II 96 II?
In the first experiment the soils were amended or not with 2 ml glucose solution (sp. act. 2992 kBq g-‘C) containing 3.3 mg N as (NH,)?SO,. In the second experiment I20 mg maize (sp. act. 940 kBq g-‘C) and 3.3 mg N as (NH,):SO, solution were added and thoroughly mixed into the soils. Both substrate amendments provided 1000 pg C g-’ soil. In each case N was added to adjust the C-to-N ratio of the added substrate to I5 (excluding the N already contained in the maize) and water. where required, to adjust the soil to 50% of WHC. The solutions were added using a speed-controlled syringe. gently mixing the solution with the soils to ensure homogeneous distribution. The soils were then incubated as described above. All measurements were done using triplicate soil portions and are expressed on an oven-dry soil basis (105’C, 24 h). Soil microbial
biomass
measurements
Biomass C measurements were made by fumigation-extraction (Vance et al., 1987b) during incubation of the soils for up to 50 days after glucose addition and for up to 100 days after addition of maize. In this technique, three amended and three unamcnded replicates of each low- and high-metal soil were fumigated with ethanol-free chloroform for 24 h at 25’C. After fumigant removal the soils wcrc extracted with 200 ml 0.5 M K,SO, for 30 min. Three amended and unamcndcd rcplicatcs of each unfumigatcd soil wcrc extracted similarly at the time furnigation commcnccd and the liltcrcd soil extracts stored at - 15°C until analysis. Organic C in the soil cxtracts was mcasurcd with a Dohrman DC80 automatic analyscr (Wu f’I al., IYYO). Briclly. 5 ml soil extract was ad&d to 5 ml potassium hcxamctaphosphatc (5%. ptl 2). The organic C in the mixture was then oxidized to CO: by reaction with pcrsulphats and ultraviolet light and the CO, cvolvcd mcusurcd by infra-red analysis. Soil microbial bio’nass C (H,) was calculated from: S, = 2.22E, where EC = [(organic C extracted from fumigated soil) minus (organic C extracted from unfumigatcd soil)] (Vance cl crl., 1987b; Wu cl al., 1990). 14C in the K:SO, soil extracts was dcterminrd by mixing I ml soil extract with 2 ml water and I5 ml RIA scintillation cocktail (Fisons plc) and counting the mixture on a scintillation counter for 5 min per sample (Vance ef ol., 1987a). “C-lab&d biomass (biomass “C) was calculated as above. Biomass
ninhpfrin-nitrogen
Ninhydrin-reactive N was mcasurcd in all soil extracts by the method of Amato and Ladd (1988). as modified by Joergcnscn and Brookcs (1990). Biomass ninhydrin-N (B,,,) W;IS cslculatcd from: B NIN= [(ninhydrin-N extracted from fumigatcd soil) minus (ninhydrin-N cxtractcd from unfumigatcd soil)]. A TP nreasuremen~s Soil ATP extraction (Jcnkinson and Oadcs, 1979) was done on the same day as the biomass C mcasurcmerits. At each time. three portions of moist soil, each
Microbial biomass dynamics in metal-contaminated containing approx. 5 g oven-dry soil, were taken from separate replicates of each treatment and weighed into 25 ml centrifuge tubes. The soils were then ultrasonified for 2 min with 25ml of extractant A [O.S M trichloroacetic acid (TCA). 0.25 M NaH,PO, and 0.1 M paraquat]. A further three soil portions of each treatment were ultrasonified with 25 ml of extractant B (extractant A containing 5 ml IO-’ M ATP). The soil suspensions were then cooled on ice, filtered through Whatman No. 42 filter papers and stored at - IS’C. ATP was measured as described by Tate and Jenkinson (1982). CO, nreasltrerrrPnts Separate jars of each soil treatment were put into stoppered I. I litres wide-mouthed bottles, containing 10 ml water and a vial containing 20 ml of I M NaOH and incubated in the dark at 25’C. The NaOH vials were changed periodically. CO,-C trapped in the NaOH was determined by HCI autotitration. “CO: was measured by mixing 2 ml aliquots of the NaOH Low-metal
soils
919
solution with 10 ml of RIA scintillation cocktail, and counting each sample for 5 min on a scintillation counter (Vance et al.. 1987a). RESULTS
AND DISCUSSION
Biomass carbon in low- and high-metal substrate addition
soils foIlowing
The initial biomass C content in the unamended low-metal soil (180 pg C g-’ soil) was twice that in the high-metal soil (90 pug C g-’ soil) and this proportional difference was maintained throughout the incubation. Glucose or maize addition significantly increased the amount of biomass C in both the lowand high-metal soils [Fig. l(a) and (b)]. The increase was highest on day 5 in both soils, remaining at about this level in the case of maize until day IO. The biomass then slowly declined until the end of the incubation. However, the net increase in total
biomass C (i.e. labelled plus unlabelled) following glucose or maize addition was larger in the low-metal High-metal
soil
(a)
soil
4 cc
25
50 days
Low-metal
soil
of
0
5
incubation High-metal
SOi1
r
Days of incubalion Fig. l(a) and (b). “C-labelled (a) and unlabelled (0) biomass C in the low- and high-metal soils during incubation following addition of (a) “C-labelled glucose and (b) “C-labelled maize. C = biomass C in unamcnded control soils: G = biomass C in glucose-amended soils; M = biomass C in maize-amended soils. LSD (P = 0.05) “C-1abellcd biomass C = 6.9, unlabellcd biomass C = 7.8 pg C g-’ soil.
(b)
K. CHANDER and P. C. BROOKES
920
than in the high-metal soil at every stage during the incubations. It should be noted that the amounts of biomass-“C formed following glucose addition were actually bigger than the increases in total biomass. This was because there was a replacement of unlabelled, native biomass C by newly synthesized biomass-“‘C derived from the added glucose. This effect did not occur when maize was added; the total increase in biomass was about equal to the amount of biomass-“C that was formed. This phenomenon had been observed previously (Brookes er al., 1990) but the reasons why glucose and plant materials cause these differences in biomass dynamics during their decomposition is not yet understood. Glucose biomass-‘JC was 152 and 103 pg C gg’ soil at day 5, declining to 64 and 49 pg C g-’ soil at day 50 in the low- and high-metal soils, respectively. The corresponding values following maize addition were I I4 and 70 jfg C g-’ soil at day 5. declining to 35 and I4 jrg C g-’ soil at day 100. This indicated that less biomass was synthesized per unit of added glucose or maize in the high-metal soil than in the low-metal soil. Overall. about 15-32% less glucose-dcrivcd and 25 -60% less maize-dcrivcd biomass-“C was formed in the high- than in the low-metal soil. These diffcrenccs wcrc in about the sumc proportions as the ditfcrcnccs in the amounts of biomass that had been ohscrvcd in the low- and high-metal ticld soils of the Woburn Market Garden Expcrimcnt (Brookcs and McGrath, 1984). In contrast, with both glucose and maize, the specific activity of the biomass W;IS higher in the high-metal than low-metal soils up to 50 days. With maize the ditfcrcncc was often not signiftcant but was much more marked with glucose [from about 10% at day 5, increasing to about 40% at day 50 (Table 2)]. This dilfcrcncc bctwccn low- and high-metal soils could have been caused by the lower initial total biomass in tho high-metal soil. Thus the labellcd-C in the newly formed biomass would have been less “diluted” by the unlabcllcd C in the original biomass. The maize-amcndcd soils wcrc incubated longer. Betwccn 75 and 100 days, the specific activities of the biomass was about 20% lower in the high- than low-metal soil (Table 2). This indicated that the biomass-“C in the high-metal soil declined faster than in the low-metal soil during further incubation.
This suggested that the biomass in the high-metal soil was both less efficient in the synthesis of new biomass from fresh substrates and that the new biomass that developed had a shorter life than the similar biomass in the low-metal soil. Changes in amounts of biomass ninhydrin-N
and high-metal
soils following
in lowsubstrate addition
Relative changes in the amounts of ninhydrin-N during incubation closely followed the changes in the amounts of biomass C in all treatments. at all times [Fig. 2(a) and (b)]. Thus there was an extremely close relationship between biomass C (S,) and biomass ninhydrin-N (B,,,). With glucose, the regression equation was B, = (25.9 + 0.24) B,,, . and with maize it was B, = (26.9 k 0.26) E,,,, both with high (r = 0.99) correlation coefficients [Fig. 2(a) and (b)]. Similarly good correlations were previously observed by Amato and Ladd (1988) and Ocio and Brookes (1990). Thus, the biomass which developed following glucose or maize addition in both the low- and high-metal soils had the same ninhydrin-N concentration as in the unamended soils. The results also indicated that the metals did not affect the ratio of C to ninhydrin-N in either the native biomass or in the biomass that developed shortly after substrate addition. C7ran~c.r in A TP contents in /OW- und high -rnetul soils
/kIlowing
substrate
addition
Initial amounts and absolute changes in amounts of ATP during the incubation of the unamcnded soils in the scparatc cxpcrimcnts with glucose and maize wcrc virtually identical although the cxpcriments wcrc done scqucntially. Thcrcforc, the values of soil ATP content (Table 3) and biomass specific rcspiration rates (Table 4) in the unamcnded low- and high-metal soils at eJch sampling time are the averages of both cxperimcnts. Changes in the ATP content of unamendcd soils during incubation closely followed the changes in biomass C. In general, the ATP contents of the unamendcd low-metal soil were, like biomass C. roughly twice those in the high-metal soil. Thus ATP concurred with biomass C and ninhydrin-N in suggesting a significantly smaller biomass in the high-metal soils. Glucose or maize addition significantly increased the ATP content of both soils
Table 2. The spccdicxuviucs of the microbial biomass in low and high-metal soils after addillon of glucose’ or mrizcb Spccitic activiucs of the microbial biomass GIUCO~C
Incubalwn period (d;lys) 5 IO 25 SO 75 IO0 LSD (P = 0.05) ’ IOOOpg
Low-mcul soil
MUX
High-metal sod (kBq g
1672 1350 II22 902 ND’ ND’
IX12 1633 I600 1331 ND< ND’
Low-melal sod
’
High-mcral solI
biomass C) 3x5 350 313 227 205 170
176.9
glucose C with specific activity of 2992 kRq g-’ C added at time 0. blOOOpg mAzc C with specific activity of 940 kBq g-’ C added at time 0.
JX 37x 325 240 157 I42 85. I
Microbial
biomass dynamics in metal-contaminated
soils
911
12(al 0 loBC-
(25.92
0.24)
BNIN
r * 0.99 /
8/ 6-
o# Low-metal
High-metal soil
soil
4Unamended
o
A
Amended
v
.
(b)
10
BC-
B s 7 0)
(27.Oi
0.27)
BNIN.
r - 0.99
/’
0
/” ,V
s
/V
1
0
50
Biomass
200
150
100 carbon
$0
300
250
g”soil)
Fig. 2(a) and (b). The correlation between biomass C and biomass ninhydrin-N in the low- and high-metal soils. with and without additions of (a) “C-labelled glucose and (b) “C-labclled maize a~ difTerent sampling times.
(Table 3). with an apparent maximum increase at day 5. Thereafter, soil ATP content dcclincd slowly in both the soils until the end of the incubations. In fact, we believe that the soil ATP contents measured in the glucose- or maize-amended soils at day 5 were erroneously large. During the early decomposition phase of the added glucose or maize (up to 5 days), the percentage recoveries of the added ATP from Extractant B were very low in both soils (3040%). Table
3. Soil
ATP
contcnt
in low-
and high-metal
Extractant B was identical to Extractant A except that it contained 25 pmol added ATP per 50 ~1 of Extractant A. During soil extraction, normally only a proportion (usually 60-80%) of the added ATP is recovered following extraction of soil with Extractant B as some of this added ATP is sorbed onto soil colioids or soil organic matter. It is assumed that native ATP behaves similarly, once it is released from the microbial cells. Thus the amount of native soil ATP extracted with Extractant A (ExA) is corrected soils with
Unamcndcd Incubation
Low-metal
and
without
addition
of glucose’
High-metal
Low-metal
or maize’
Maize
GIUCOX
High-metal
Low-metal
High-metal
period (nmol
(days)
ATP
g
’ roil)
0
2.24
I.14
-
-
-
-
5
2.24
I.13
(6.87)’
(3.37)’
(6.47)’
(3.12)’ 2.06
IO
2.12
I .08
3.31
I .86
4.26
2s
2.15
1.06
3.02
1.79
3.20
I .79
50
2.00
0.98
2.54
1.29
2.94
I .37
75
1.85
0.94
NDb
ND”
2.36
I.21
100
1.87
0.86
NDb
NDb
2.21
0.95
LSD
with
0.192
0.121
(P - 0.05)
‘Amended
glucose
‘ND
-
‘For
an cxplanarion
or maize
as in Table
2.
not determined. of these anomalously
high values
see text.
0. I38
K. CHASFDER and P. C. B~oorcfs
9”__
Table 4. Btomass soccific respiration rakes in low- and high-metal soils following addition of &JCOSC’or mai& Biomass soecific resoiration raw? Unamenjed Incubatmn perrod ldavsl o-5 5-10 l&25 2>50 50-75 7SlMl LSD {P = 0.05)
Glucose
Low-metal
High-m&al
35 33 31 30 28 25
51 52 56 41 35 33
Low-metal
Maize
High-metal
tmn CO.-C a-’ biomass C day“ 463 59 59 30 ND” ND*
3.1
764 86 86 38 ND’ ND’
Low-metal
High-melal
250 109 44 26 26 2s
520 244 86 42 43 37
)
4.3
4.1
‘Amended with plucox or maize as in TAble 2. b610mass rpcc~fic resplratton rates were calculated from lolai CO& evolved during the incubation within the dltkent time intenals dwided by the averages of the amounts of bmmass carbon measured at the first and lasl dav of each inrenal and dwldcd bv the number of days in each interval period. rcspec11vely. ‘ND = not determmcd.
by reference to the percentage recovery of ATP obtained with Extractant B (ExB). to account for its incomplete extraction, from the relationship: 25 (soil ATP extracted with ExA) soil ATP =
(soil ATP extracted with ExB - soil ATP extracted with ExA)
estimate of biomass in uncontaminated and metalcontaminated soils and that the metals do not change the biomass ATP concentration. Cironges in CO_, ecolution in low - and high -metal soils
’
After day 5. the recoveries of added ATP from Extractant B were as cxpccted (i.e. 6G30% of the amount added). We hnvc observed anomalously low percentage rccovcrics of addod ATP in Extrrrctlrnt B scvcr~l times in other (unpublished) experiments shortly after substrate addition and it does appear to be a real c&ct. As yet. we cannot olfcr a satisfactory explanation, altll~~u~h it is being studied further in our I;rbor;ttory (MGa Di Nobili, personal communication). It does strongly suggest that soil ATP contents (corrected for individual recoveries of added ATP) measured in the glucose- or maize-amended soils, were greatly ovorcstimatcd up to day 5. When data obtained af day 5 were omitted, a very close linear relationship between biomass C and ATP w;ts obtained. irrespective of whsther the soils had rcceived glucose or maize [Fig. 3(a) and (b)]. If the soil ATP contents obtained at day 5 were corrected with the much higher percentage recoveries of ATP obtained at later sampling dates. the soil ATP contents measured at day 5 also fitted the regression well. This also suggested that these measured percentage recoveries of ATP were in error. between For glucose. the regression equation biomass C and soil ATP was: E, = (79.6 & 0.71) ATP. and. for maize: S, = (80.4 k0.88) ATP, both with high (r = 0.99) correlation cocficients {Fig. 3(a) and (b)]. Thus the biomass in both the low- and highmetal soils, with and without added substrates, had the same ATP concentration. The biomass ATP concentration meaned over both soils with and without added substrates (omitting the values obtained in amended soils at day 5) was 12.3 + 0.16pmol ATP g-’ biomass carbon. This value is close to that reported by Jenkinson (1988) of I I .7 ~trnol ATP g-’ biomass carbon, obtained from a large number of unamcnded soils taken from the literature and Ocio and Brookes (1990) for unamended soils and soils containing recently added wheat straw. This again suggests that ATP measurements provide a valid
following
substrate addition
More total and labelled CO, was evolved from the high-metal than the low-metal soil during the early, rapid decomposition phase following substrate addition. Once the tlush of decomposition was over (at 5 days for glucose and 50 days for maize). the mtes of CO: evolution declined to lower values than in the low-metal soil [Fig. 4(n) and (b)]. The amounts of total CO: evolved from the unamendcd low-metal soils wcrc l5-20% larger than the amounts cvolvcd from the high-rnet~l soil over the entire incubation period (data not given). In contrast, the specific respiration ratio [(biomass specific respiration rate in high-metal soil)/(spccific respiration rate in low-metal soil)] was about I.5 in the unamcndcd soils up to about day 25. After this time the difference in respiration rates became slightly less, so that between days 75 and 100 the specific respiration ratio was about 1.3 (Table 4). Following addition of glucose, the specific respiration rates were many times faster than in the unamcndcd soils betwccn days 0 and 5. Thereafter, they declined rapidly. However, the specific respiration ratios in the glucose-amended soils were similar at different sampling dates throughout the incubation. Thus, between days 0 and 25 the ratio was about 1.5, declining to about I.3 between days 25 and 50. This biomass specific respiration ratio between the glucose amended highand low-metal soils was therefore quite similar to the ratio between the unamended high- and low-metal soils. Biomass specific respiration rates in the maizeamended, high-metal soil were also consistently faster than in the maize-amended low-metal soil. Between days 0 and 25, the specific respiration ratio was about 2, declining to about 1.5 between days 25 and 100 (Table 4). These results therefore show that the smaller biomass in the high-metal soil (both substrate amended and unamended) had a faster specific respiration rate than the larger biomass in the low-metal soil. As a consequence of this, the ratio [(biomass lJC)/(respired “CO,)] was about 4@-50% less in the
Microbial
biomass dynamics in met&contaminated
8G (79.7
923
soils
f 0.7 1)ATP. 8
r * 0.99
EC*
35 ‘: 0,
(8’3.4!
0.89)ATP.
r * 0.99
84 .E n3 2 2
@D*.CYvG
;
i
x/@f:
0
i 50
100 BiomxK+
t50 carbon
200
250
305
(pg 9” soil)
The correlation between biomass C and soil ATP contents in the low- and high-metal soils. with and without addition of (a) “C-labellcd glucose and (b) “C4abelled tnaizc at difkrent sampling times. Note: The points 8. Q were from day 5 and were not included in the regression analysis (see text). Fig.
gfucosr-
3(a)
and
(b).
or maize-amen&d high-metal soifs during &composition phase (Q-5 days) (Tabfc 5). II is. however, probnbfy significant that the sum of biomass “C itnd rcspircd “CO, in the lowand high-metal soil Following addition of glucose (636 and 65Opg Cg _’ soil, rcspcctivefy) and maize (395 and 4% pg C g-l soil, r~spectjvely) were so cfose during the first 5 days following substrate addition. This the early
implies
in both the high- and abilities to metabolize the added gfucose-C or maize-C. However. much more of this C was converted t5 new biomass in the low- than in the high-metal soil. Conversely, much more of the available C fram the added substrate was evolved as C02-C from the biomass in the high-metaf than in the low-metal soil. low-met4
that
soil
the
biomass
had similar
Fig. J(a) and (h). Total and “C-lab&d CO: evolved from the low- and high-metal soils during incubation (4 “C4abclled glucose and (b) “C-labelled maize. The overall analytical coefficient of variation of all analyses is less than 5%.
K. CHASDER and P. C. BROOM TJblc
5. Biomass wds
“C.
dunng
“CO:
cvolvcd
the first 5 day
and the ratm
[(biomass
following
of incubation
“Cl (‘%IO: cvolvcdl~ III lowdddmon of “C-l.hclled glucox’ Sum
Btomass
“C
Amendment
LIlP c
GIUCOSC
Low-metal
soil
High-metal Maize
‘Amended bRatto The
with
sod
Low-merat
soil
High-metal
so11
&~ose
[(biomass
owall
“CO:
or maxe
“C,respired
coefficients
‘*CO:
P
of biomass
plus cwlwd
“C
“CO:
RJU0~
’ SOlI)
152
484
636
0.31
103
S?
650
0.19
I I4
181
395
0.40
70
336
JO6
0.21
as in Table
of varratxon
e\okd
Jnd hlgh.mctal or malzc’
2.
)I. for
biomass
“C
This increased diversion of C from biosynthesis to respired CO2 in the high-metal soil suggests that the metals are causing stress to the microbial biomass possibly by the mechanisms discussed in the Introduction. The stress is reflected in the higher specific respiration rates and consequently smaller amounts of substrate C incorporated into the microbial biomass in the high-metal soils. Recently, smaller biomasses were also found in metal-contaminated soils from other field experiments which rcccivcd past applications of metalcontaminated sewage-sludge. Chandcr and Brookcs (1991) reported that both Cu and Zn separately at 2-3 times current EC pcrmittcd limits decreased the amount of biomass by about 40% in both a sandyloam and silty-loam soil. However. the metal concentrations in thcsc soils were appreciably higher than at Wohurn. WC thorcforc suggest that our laboratory results may help to explain the previous finding of smaller b&masses in the high-metal soils of the Woburn Market Garden Experiment (Brookes and McGrath. 19x4). and thcso ctT’ccts may well operate at cvcn larger soil mctnl concentrations. However, extrapolation of results obtained under controlled laboratory conditions to those obtained from the field must always he done with caution. In particular, the biomass in the licld will receive small intermittent pulses of fresh substrates, such as roots, root exudates and plant rcsiducs. In contrast, in this work, both substrates were supplied as a single, large pulse, equivalent to 3.5 t C ha -’ in the O-23 cm plough layer. Also, while the mechanisms we have observed in this laboratory study probably also operate under field conditions, there is an ~tddition~tl possible mechanism. This is that the metals also exert direct toxic ctrects upon the plants thcmsolvcs, leading, in turn. to decreased inputs of plant-derived substrates and, consequently, to a smaller biomass. This possibility also requires investigation. Ac~~n,~l~,~f~r,r~t~,~~~.~ -K. Chandcr thanks the Commonwealth Commission and The British Council U.K. for financial support. We also thank R. Martens for supplying the “C-labellcd maize. S. P. McGrath for practical help and P. J. Harris, D. S. Jenkinson. S. P. McGrath and A. Wild for useful discussion.
REFERENCES
Amato M. and Ladd J. N. (1988) Assay for microbial biomass based on ninhydrin-reactive nitrogen in extracts of fumigated soils. Soit Biatogy & Biochemisrry 20, 107-I 1.4.
and
“CO,
evolved
are ‘.I
and
Z 6*..
reqxcwely.
Babich H. and Stotzky G. (lY77) Sensitivity of various bacteria. including actinomycetes and fungi. to cadmium and the influence of pH on sensitivity. Applied and Encironmennral Microbiology 33. 68 I-695. Babich H. and Stotzky G. (1982) Toxicity of nickel to micro-organisms in soil: Influence of some physic~hemicai characteristics. Enc~r(fnnr~,nfat Pollaiian 29& 303-31s. Bhattacherjee 1. W. (1986) Etfect of cadmium and zinc on microbtai adhesion, growth and metal uptake. Buttefin oj &nrironmenfal Cot~rtrmma/rcm and Toxicology 36, 396300. Bremncr J. M. (1985) Total nitrogen. In &l&o& of Suit Anaf.r.ris Foi. 2 (C. A. Black. Ed.), pp. 1149-l 178. Amcrican Society of Agronomy. Madison, Wis. Brookcs P. C. and McGrnth S. P. (19X.4) Effects of metal toxicity on the site of the soil microbial biomass. lournul 0P Soil Scic*rux* 35, 341 346, Brookcs P. C.. 0% J. A. ;rnd Wu J. (IWO) The soil microbial biomass: its mciksurcmcnt, properties and role in soil nitrogen and carbon Jyn.mics following substrate incorp~~~Iti~)n. Soil *~fr~rttor,~rjtrr.rttrs 35, 3Y -5 1. Chandcr K. and Brookcs P. (‘. f IYYI) Etfccts of heavy mctnls from past :Ipphc;ltions of scwaye sludge on microblal biomass and organic m;lttsr accumulation in a sandy loam and a silty loam U.K. soil. Soil Uiotqy & Bioc/wmi.srr), 23. 927 932. Coilins V. E. and Stotzky G. (IYxO) Factors atfccting the toxicity of heavy met;rls to microbes. In itletuf 1tlp1.srind &creria (T. J. Brvcridgc and R. J. Doyle. Eds), pp. 31-90. Wiley. New York. Commission of ths European Communities (19X6) Council Directive on the protection of the environment. and in particular of the soil, whrn sewage sludge is used in agriculture. Oficiut ./ournal a~‘rlte European Cammunifies L181. (Annex IA) IO. Duxbury T. (198 I) Toxicity of heavy metals to soil bacteria. FE.%I.S ,~ficrohiataqy f.efrer.s t I, t I 7-220. Gadd 6. M. and Grifliths A. J. (1978) Microorganisms and heavy metal toxicity. Microhiut Ecology -5, 303317. Jenkinson D. S. (1988) Dctcrmination of microbial biomass carbon and nitrogen in soil. In Adrance.~ in Nirragen C_rcting in A.tyicuirttrat &cr~.s.wrwx (J. R. Wilson, Ed.), pp. 368-386. Comrn~~nw~Ilth floricultural Bureau international. Wallingford. Jenkinson D. S. and Oadcs J. M. (1979) A method for measuring adsnosine triphosphate in soil. &it Biology & Biochemisfr~ II. 193-199. Jocrgenscn R. G. and Brookes P. C. (1990) Ninhydrinreactive nitrogen measurements of microbial biomass in 0.5 in K,SO, soil extracts. Ser.’ Siatqy & Etoc~ernjsl~~ 22, 1023-1027. Johnston A. E. and Wcdderburn R. W. M. (1975) The Woburn Market Garden Experiment. 194249. I. A history of the experiment, details of the treatments and the yields of the crops. Rothamsred E?rperimentai Starion Repor/Jb IY74, Part 2. pp. 79.-101.
Microbtal
biomass
dynamics
Kalembasa S. J. and Jenkinson D. S. (1973) A comparative study of titrimetric and gravimetric methods for the determination of organic carbon in soil. hurna/ qf rhr Science of Food and Agriculrure 24. 1085-1090. Killham K. (1985) A physiologtcal determination of the impact of environmental stress on the activity of microbial biomass. Erwironmenrul Pollution 38. 283-294. Killham K. and Firestone M. K. (1984) Salt stress control of intracellular solutes in streptomycetes indigenous to saline soils. Applied and Erwironmenral Microbiology 47. 30 l-306. McGrath S. P. and Cunliffe C. H. (1985) A simplified method for the extraction of the metals Fe. Zn. Cu. Ni. Cd. Pb, Cr. Co and Mn from soils and sewage sludges. Joumul of rhe Science of Fuud and Agriculture 36. 794-798. 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 develoos. Soil Bioluar & Biochemistry 22. 685-694. Rachlin J. W.. Jenson T. E., Baxter M. and Jani V. (1982) Utihzation of morphometric analysis in evaluating response of Plecronema boryanum (Cyanophyceae) to exposure to eight heavy metals. Archires of Environmenfal Con~aminafion & Toxicology I I. 323-333. Y.
in metal-contaminated
soils
925
Rich S. and Horsfall J. G. (1963) Fungicides as metabolic inhibitors. In Metabolic Inhibirurs (R. M. Hochster and I. H. Quastel. Eds). Vol. 2. pp. 263-284. Academic Press, New York. Siegel S., Keller P., Galun M.. Lehr H.. Siegel B. and Galun E. (1986) Biosorption of lead and chromium by Penicillium preparations. U’ufer, Air & Soil Polkion 27. 69-75. Tate K. R. and Jenkinson D. S. (1981) Adenosine triphosphate (ATP) and microbial biomass in soil: etfects of storage at different temperatures and at different moisture levels. Communications in Soil Science and Plum .Analuis 13. 899-908. Vance E. D.. Brookes P. C. and Jenkinson D. S. (1987a) Microbial biomass measurements in forest soils: determination of k; values and tests of hypotheses to explain the failure of the chloroform fumigation-incubation method in acid soils. Soil Bioloxr & Biochemurrv 19. 689696. Vance E. D.. Brookes P. C. and Jenkinson D. S. (1987b) An extraction method for measuring microbial biomass C. Soil Biology & Biochemisyv 19, 703-707. Wu J.. Joergensen R. G.. Pommerening 8.. Chaussod R. and Brookes P. C. (1990) Measurement of soil microbial biomass C by fumigation-extraction-an automated procedure. Soil Biology & Biochemisyv 22, 1167-l 169.
Budtern
Vol. 13. No
0038-0717.91 $3.00+
IO. pp. 917-925. 1991
0.00
Copyright C 1991 Pergamon Press plc
Pnntcd m Great Bntam. All nghts reserved
MICROBIAL BIOMASS DYNAMICS DURING THE DECOMPOSITION OF GLUCOSE AND MAIZE IN METAL-CONTAMINATED AND NON-CONTAMINATED SOILS K. CHASDER* Soil Science Department.
AFRC
and P. C. BRooKEst
Institute of Arable Crops Research. Rothamsted Harpenden. Herts AL5 2JQ. U.K.
Experimental
Station.
(Accepted 5 May 1991) Summary-Metal-contaminated soils (produced by past long-term applications of contaminated sewagesludge) from the Woburn Market Garden Field Experiment were previously shown to contain only about half the amounts of microbial biomass as other soils from the experiment which received farmyard manure during the same period. In some cases. the amounts of biomass in the metal-contaminated soils were even smaller than in other soils from the experiment which received inorganic fertilizer throughout. It is possible that the metals were causing decreased efficiency of substrate utilization by the microbial biomass. leading. in turn. to a smaller microbial population. This was investigated in a laboratory glucose and “C-labelled experiment by adding “C-labelled maize shoots (maize) separately to a metal-contaminated and a non-contaminated soil from the field experiment. Microbial biomass C. ninhydrin-N. soil ATP content and CO? evolution were measured during the next 50 days following glucose addition and 100 days following maize addition in both soils. The biomass formed following addition of glucose or maize was consistently smaller in the metal-contaminated soil throughout the incubations. Overall. about I5 -32% less glucose-derived and 25-60X less maize-derived biomass was formed in the mct;ll-contamin;ttcd soil. In contrast, more CO& was cvolvcd from the metal-contaminated soil than from the non-cont;lmin;~tcd soil. This suggests that the biomass in the metal-contaminated soil was less cllicicnt in the uttli~;ttion of substrates for biomass synthesis. It is suggested that this may be a major reason for the smaller biomass in the mct&contaminatcd Woburn soils.
IN’l’WOI)tiC-TION
when micro-organisms cxpericnccd such cnvironmcntal strcsscs. including heavy metals. Large concentrations of heavy metals arc harmful to the growth, morphology and metabolism of microorganisms in oifro (e.g. Babich and Stotzky, 1977, 1982; Bhattachcrjee, 1986; Duxbury. 1981; Rachlin rr 01.. 1982; Siegel er 01.. 1986). The binding of metals onto sites on the cell wall may lead to changes in the morphology of the ccl1 surface, and disrupt normal functioning (Collins and Stotzky, 1989). Heavy metals may also denature proteins (e.g. Gadd and Griffiths, 1978) or destroy the integrity of cell mcmbranes, allowing mctabolites to leak out (Rich and Horsfall. 1963). Organisms living in metal-contaminated environments may therefore need to expend more energy to survive than if they lived in less stressful situations. Thus it might be expected that the cfficicncy of conversion of fresh substrates into new soil microbial biomass may be less cfhcicnt, respiration may be faster and the rate of microbial turnover increased at clcvatcd soil metal contents. If these factors opcratcd, they could help to explain the smaller biomasscs that have been observed in metal-contaminated soils. We therefore have dctcrmincd if heavy metals exert such direct toxic effects on the microbial biomass, using soils from the Woburn Expcrimcnt. This was done by adding “C-labelled glucose and “C-labellcd maize shoots to uncontaminated and metal-contaminated soils and measuring the synthesis of biomass
In 1984, soils from the Woburn Market Garden Expcrimcnt, which last received metal-contaminated scwagc-sludge 23 yr previously (Johnston and Wcddcrburn. 1975) were reported to contain only about half the amounts of microbial biomass found in other soils from the cxpcrimcnt which rcccivcd farmyard manure during the same period (Brookcs and McGwth. 1984). In some cases the biomass in the soils contaminated with heavy metals (e.g. Cu. Ni, Cd and Zn) was even smaller than in soils that had received inorganic fcrtilizcr throughout the experimcnt. The mechanisms producing the smaller biomasses in the metal-contaminated soils [now containing Cu. Ni and Zn at about the EC limit (Commission of the European Communities. 1986). and Cd at about two to three times above it] were not then investigated. However, it is known that cnvironmcntal stresses (such as extrcmcs of pH. tcmpcraturc, salinity or metal content) perturb the division of cncrgy bttwecn growth and maintcnancc in micro-organisms. For example. Killham (1985) and Killham and Fircstonc (1984) reported increased diversion of carbon from biosynthesis to maintcnancc cncrgy rcquircmcnts -___I_
-__-
*Permanent address: Dcpartmcnt of Soil Science Haryana Agricultural University. Hisar. India. tAuthor for correspondence. 917
K. CHANDER and P. C. B~oorcas
918
and CO,-C evolved as the substrates decomposed under laboratory conditions. We used biomass C and biomass ninhydrin-N measurements (by fumigationextraction) and biomass ATP measurements because these methods have been shown (Ocio and Brookes. 1990) to provide reliable biomass estimates in unamended soils and in soils containing recent inputs of readily-decomposable substrates.
MATERIALS
AND METHODS
Soils Soils were sampled to plough depth (0-23cm) in October 1988 from plot No. 2. which had received farmyard manure from 1942 to 1967 (low-metal soil) and plot No. 39, which had received metal-contaminated sewage-sludge from 1942 to 1961 (high-metal soil) of the Woburn Market Garden Experiment. Since then, both soils have received inorganic fertilizer only. The soils were stored moist at S’C until use, after removing plant material, stones and visible soil fauna. They were then sieved (< 2 mm). adjusted to 40% of water holding capacity (WHC) and were equilibrated for 7 days at 25 C over water and soda lime in air-tight metal bins before use. Other portions of soils were air-dried and then finely ground in a Tcma mill bcforc chemical analysis. A brief description of the soil propcrtics is given in Table I.
Soil pH was mcasurcd with a glass clcctrodc using a I : 2.5 soil-to-water ratio. Total soil N was mcasurcd by Kjcldahl dig&on (Brcmncr. IY65) and organic C in plant material and soils by dichromatc digestion (Kalcmbasa and Jcnkinson, lY73). Total metals wcrc dctcrmincd after digestion of the soils with 4: I (v/v) HCI: HNO, (McGrath and Cunlilfc, 19x5). Soil treatments Two separate laboratory experiments were done, one using “C-labcllcd glucose and the other using uniformly “C-labclIcd maize shoots (maize) as substratc. The maize was oven-dried at 35°C for 2 days and then lincly ground (< 160 pm) before use. Three sets of moist low- and high-metal soil (each containing 50g soil on an oven-dry basis) were weighed into 100 ml jars. The first set was used to measure soil microbial biomass C and biomass ninhydrin-N. the second to measure evolved CO, and the third for ATP measurements.
Table
I. Analysis of soils from the Wohurn Erpcnment
PII Organic C (%) Total N (%) Clay (%I TCXllKC Tuotul metal (pg * Zll
CU Ni Cd Cr Pb
Market
Garden
Low-metal 5011
High-mctal soil
7.06
7.06 I .6? 0.16 9.0 Loamy-sand
I.25 0.13 90 Loamy-tand
concmrrorion
’ soil)
107 26 16 I.4 4n 42
419 12s M II 96 II?
In the first experiment the soils were amended or not with 2 ml glucose solution (sp. act. 2992 kBq g-‘C) containing 3.3 mg N as (NH,)?SO,. In the second experiment I20 mg maize (sp. act. 940 kBq g-‘C) and 3.3 mg N as (NH,):SO, solution were added and thoroughly mixed into the soils. Both substrate amendments provided 1000 pg C g-’ soil. In each case N was added to adjust the C-to-N ratio of the added substrate to I5 (excluding the N already contained in the maize) and water. where required, to adjust the soil to 50% of WHC. The solutions were added using a speed-controlled syringe. gently mixing the solution with the soils to ensure homogeneous distribution. The soils were then incubated as described above. All measurements were done using triplicate soil portions and are expressed on an oven-dry soil basis (105’C, 24 h). Soil microbial
biomass
measurements
Biomass C measurements were made by fumigation-extraction (Vance et al., 1987b) during incubation of the soils for up to 50 days after glucose addition and for up to 100 days after addition of maize. In this technique, three amended and three unamcnded replicates of each low- and high-metal soil were fumigated with ethanol-free chloroform for 24 h at 25’C. After fumigant removal the soils wcrc extracted with 200 ml 0.5 M K,SO, for 30 min. Three amended and unamcndcd rcplicatcs of each unfumigatcd soil wcrc extracted similarly at the time furnigation commcnccd and the liltcrcd soil extracts stored at - 15°C until analysis. Organic C in the soil cxtracts was mcasurcd with a Dohrman DC80 automatic analyscr (Wu f’I al., IYYO). Briclly. 5 ml soil extract was ad&d to 5 ml potassium hcxamctaphosphatc (5%. ptl 2). The organic C in the mixture was then oxidized to CO: by reaction with pcrsulphats and ultraviolet light and the CO, cvolvcd mcusurcd by infra-red analysis. Soil microbial bio’nass C (H,) was calculated from: S, = 2.22E, where EC = [(organic C extracted from fumigated soil) minus (organic C extracted from unfumigatcd soil)] (Vance cl crl., 1987b; Wu cl al., 1990). 14C in the K:SO, soil extracts was dcterminrd by mixing I ml soil extract with 2 ml water and I5 ml RIA scintillation cocktail (Fisons plc) and counting the mixture on a scintillation counter for 5 min per sample (Vance ef ol., 1987a). “C-lab&d biomass (biomass “C) was calculated as above. Biomass
ninhpfrin-nitrogen
Ninhydrin-reactive N was mcasurcd in all soil extracts by the method of Amato and Ladd (1988). as modified by Joergcnscn and Brookcs (1990). Biomass ninhydrin-N (B,,,) W;IS cslculatcd from: B NIN= [(ninhydrin-N extracted from fumigatcd soil) minus (ninhydrin-N cxtractcd from unfumigatcd soil)]. A TP nreasuremen~s Soil ATP extraction (Jcnkinson and Oadcs, 1979) was done on the same day as the biomass C mcasurcmerits. At each time. three portions of moist soil, each
Microbial biomass dynamics in metal-contaminated containing approx. 5 g oven-dry soil, were taken from separate replicates of each treatment and weighed into 25 ml centrifuge tubes. The soils were then ultrasonified for 2 min with 25ml of extractant A [O.S M trichloroacetic acid (TCA). 0.25 M NaH,PO, and 0.1 M paraquat]. A further three soil portions of each treatment were ultrasonified with 25 ml of extractant B (extractant A containing 5 ml IO-’ M ATP). The soil suspensions were then cooled on ice, filtered through Whatman No. 42 filter papers and stored at - IS’C. ATP was measured as described by Tate and Jenkinson (1982). CO, nreasltrerrrPnts Separate jars of each soil treatment were put into stoppered I. I litres wide-mouthed bottles, containing 10 ml water and a vial containing 20 ml of I M NaOH and incubated in the dark at 25’C. The NaOH vials were changed periodically. CO,-C trapped in the NaOH was determined by HCI autotitration. “CO: was measured by mixing 2 ml aliquots of the NaOH Low-metal
soils
919
solution with 10 ml of RIA scintillation cocktail, and counting each sample for 5 min on a scintillation counter (Vance et al.. 1987a). RESULTS
AND DISCUSSION
Biomass carbon in low- and high-metal substrate addition
soils foIlowing
The initial biomass C content in the unamended low-metal soil (180 pg C g-’ soil) was twice that in the high-metal soil (90 pug C g-’ soil) and this proportional difference was maintained throughout the incubation. Glucose or maize addition significantly increased the amount of biomass C in both the lowand high-metal soils [Fig. l(a) and (b)]. The increase was highest on day 5 in both soils, remaining at about this level in the case of maize until day IO. The biomass then slowly declined until the end of the incubation. However, the net increase in total
biomass C (i.e. labelled plus unlabelled) following glucose or maize addition was larger in the low-metal High-metal
soil
(a)
soil
4 cc
25
50 days
Low-metal
soil
of
0
5
incubation High-metal
SOi1
r
Days of incubalion Fig. l(a) and (b). “C-labelled (a) and unlabelled (0) biomass C in the low- and high-metal soils during incubation following addition of (a) “C-labelled glucose and (b) “C-labelled maize. C = biomass C in unamcnded control soils: G = biomass C in glucose-amended soils; M = biomass C in maize-amended soils. LSD (P = 0.05) “C-1abellcd biomass C = 6.9, unlabellcd biomass C = 7.8 pg C g-’ soil.
(b)
K. CHANDER and P. C. BROOKES
920
than in the high-metal soil at every stage during the incubations. It should be noted that the amounts of biomass-“C formed following glucose addition were actually bigger than the increases in total biomass. This was because there was a replacement of unlabelled, native biomass C by newly synthesized biomass-“‘C derived from the added glucose. This effect did not occur when maize was added; the total increase in biomass was about equal to the amount of biomass-“C that was formed. This phenomenon had been observed previously (Brookes er al., 1990) but the reasons why glucose and plant materials cause these differences in biomass dynamics during their decomposition is not yet understood. Glucose biomass-‘JC was 152 and 103 pg C gg’ soil at day 5, declining to 64 and 49 pg C g-’ soil at day 50 in the low- and high-metal soils, respectively. The corresponding values following maize addition were I I4 and 70 jfg C g-’ soil at day 5. declining to 35 and I4 jrg C g-’ soil at day 100. This indicated that less biomass was synthesized per unit of added glucose or maize in the high-metal soil than in the low-metal soil. Overall. about 15-32% less glucose-dcrivcd and 25 -60% less maize-dcrivcd biomass-“C was formed in the high- than in the low-metal soil. These diffcrenccs wcrc in about the sumc proportions as the ditfcrcnccs in the amounts of biomass that had been ohscrvcd in the low- and high-metal ticld soils of the Woburn Market Garden Expcrimcnt (Brookcs and McGrath, 1984). In contrast, with both glucose and maize, the specific activity of the biomass W;IS higher in the high-metal than low-metal soils up to 50 days. With maize the ditfcrcncc was often not signiftcant but was much more marked with glucose [from about 10% at day 5, increasing to about 40% at day 50 (Table 2)]. This dilfcrcncc bctwccn low- and high-metal soils could have been caused by the lower initial total biomass in tho high-metal soil. Thus the labellcd-C in the newly formed biomass would have been less “diluted” by the unlabcllcd C in the original biomass. The maize-amcndcd soils wcrc incubated longer. Betwccn 75 and 100 days, the specific activities of the biomass was about 20% lower in the high- than low-metal soil (Table 2). This indicated that the biomass-“C in the high-metal soil declined faster than in the low-metal soil during further incubation.
This suggested that the biomass in the high-metal soil was both less efficient in the synthesis of new biomass from fresh substrates and that the new biomass that developed had a shorter life than the similar biomass in the low-metal soil. Changes in amounts of biomass ninhydrin-N
and high-metal
soils following
in lowsubstrate addition
Relative changes in the amounts of ninhydrin-N during incubation closely followed the changes in the amounts of biomass C in all treatments. at all times [Fig. 2(a) and (b)]. Thus there was an extremely close relationship between biomass C (S,) and biomass ninhydrin-N (B,,,). With glucose, the regression equation was B, = (25.9 + 0.24) B,,, . and with maize it was B, = (26.9 k 0.26) E,,,, both with high (r = 0.99) correlation coefficients [Fig. 2(a) and (b)]. Similarly good correlations were previously observed by Amato and Ladd (1988) and Ocio and Brookes (1990). Thus, the biomass which developed following glucose or maize addition in both the low- and high-metal soils had the same ninhydrin-N concentration as in the unamended soils. The results also indicated that the metals did not affect the ratio of C to ninhydrin-N in either the native biomass or in the biomass that developed shortly after substrate addition. C7ran~c.r in A TP contents in /OW- und high -rnetul soils
/kIlowing
substrate
addition
Initial amounts and absolute changes in amounts of ATP during the incubation of the unamcnded soils in the scparatc cxpcrimcnts with glucose and maize wcrc virtually identical although the cxpcriments wcrc done scqucntially. Thcrcforc, the values of soil ATP content (Table 3) and biomass specific rcspiration rates (Table 4) in the unamcnded low- and high-metal soils at eJch sampling time are the averages of both cxperimcnts. Changes in the ATP content of unamendcd soils during incubation closely followed the changes in biomass C. In general, the ATP contents of the unamendcd low-metal soil were, like biomass C. roughly twice those in the high-metal soil. Thus ATP concurred with biomass C and ninhydrin-N in suggesting a significantly smaller biomass in the high-metal soils. Glucose or maize addition significantly increased the ATP content of both soils
Table 2. The spccdicxuviucs of the microbial biomass in low and high-metal soils after addillon of glucose’ or mrizcb Spccitic activiucs of the microbial biomass GIUCO~C
Incubalwn period (d;lys) 5 IO 25 SO 75 IO0 LSD (P = 0.05) ’ IOOOpg
Low-mcul soil
MUX
High-metal sod (kBq g
1672 1350 II22 902 ND’ ND’
IX12 1633 I600 1331 ND< ND’
Low-melal sod
’
High-mcral solI
biomass C) 3x5 350 313 227 205 170
176.9
glucose C with specific activity of 2992 kRq g-’ C added at time 0. blOOOpg mAzc C with specific activity of 940 kBq g-’ C added at time 0.
JX 37x 325 240 157 I42 85. I
Microbial
biomass dynamics in metal-contaminated
soils
911
12(al 0 loBC-
(25.92
0.24)
BNIN
r * 0.99 /
8/ 6-
o# Low-metal
High-metal soil
soil
4Unamended
o
A
Amended
v
.
(b)
10
BC-
B s 7 0)
(27.Oi
0.27)
BNIN.
r - 0.99
/’
0
/” ,V
s
/V
1
0
50
Biomass
200
150
100 carbon
$0
300
250
g”soil)
Fig. 2(a) and (b). The correlation between biomass C and biomass ninhydrin-N in the low- and high-metal soils. with and without additions of (a) “C-labelled glucose and (b) “C-labclled maize a~ difTerent sampling times.
(Table 3). with an apparent maximum increase at day 5. Thereafter, soil ATP content dcclincd slowly in both the soils until the end of the incubations. In fact, we believe that the soil ATP contents measured in the glucose- or maize-amended soils at day 5 were erroneously large. During the early decomposition phase of the added glucose or maize (up to 5 days), the percentage recoveries of the added ATP from Extractant B were very low in both soils (3040%). Table
3. Soil
ATP
contcnt
in low-
and high-metal
Extractant B was identical to Extractant A except that it contained 25 pmol added ATP per 50 ~1 of Extractant A. During soil extraction, normally only a proportion (usually 60-80%) of the added ATP is recovered following extraction of soil with Extractant B as some of this added ATP is sorbed onto soil colioids or soil organic matter. It is assumed that native ATP behaves similarly, once it is released from the microbial cells. Thus the amount of native soil ATP extracted with Extractant A (ExA) is corrected soils with
Unamcndcd Incubation
Low-metal
and
without
addition
of glucose’
High-metal
Low-metal
or maize’
Maize
GIUCOX
High-metal
Low-metal
High-metal
period (nmol
(days)
ATP
g
’ roil)
0
2.24
I.14
-
-
-
-
5
2.24
I.13
(6.87)’
(3.37)’
(6.47)’
(3.12)’ 2.06
IO
2.12
I .08
3.31
I .86
4.26
2s
2.15
1.06
3.02
1.79
3.20
I .79
50
2.00
0.98
2.54
1.29
2.94
I .37
75
1.85
0.94
NDb
ND”
2.36
I.21
100
1.87
0.86
NDb
NDb
2.21
0.95
LSD
with
0.192
0.121
(P - 0.05)
‘Amended
glucose
‘ND
-
‘For
an cxplanarion
or maize
as in Table
2.
not determined. of these anomalously
high values
see text.
0. I38
K. CHASFDER and P. C. B~oorcfs
9”__
Table 4. Btomass soccific respiration rakes in low- and high-metal soils following addition of &JCOSC’or mai& Biomass soecific resoiration raw? Unamenjed Incubatmn perrod ldavsl o-5 5-10 l&25 2>50 50-75 7SlMl LSD {P = 0.05)
Glucose
Low-metal
High-m&al
35 33 31 30 28 25
51 52 56 41 35 33
Low-metal
Maize
High-metal
tmn CO.-C a-’ biomass C day“ 463 59 59 30 ND” ND*
3.1
764 86 86 38 ND’ ND’
Low-metal
High-melal
250 109 44 26 26 2s
520 244 86 42 43 37
)
4.3
4.1
‘Amended with plucox or maize as in TAble 2. b610mass rpcc~fic resplratton rates were calculated from lolai CO& evolved during the incubation within the dltkent time intenals dwided by the averages of the amounts of bmmass carbon measured at the first and lasl dav of each inrenal and dwldcd bv the number of days in each interval period. rcspec11vely. ‘ND = not determmcd.
by reference to the percentage recovery of ATP obtained with Extractant B (ExB). to account for its incomplete extraction, from the relationship: 25 (soil ATP extracted with ExA) soil ATP =
(soil ATP extracted with ExB - soil ATP extracted with ExA)
estimate of biomass in uncontaminated and metalcontaminated soils and that the metals do not change the biomass ATP concentration. Cironges in CO_, ecolution in low - and high -metal soils
’
After day 5. the recoveries of added ATP from Extractant B were as cxpccted (i.e. 6G30% of the amount added). We hnvc observed anomalously low percentage rccovcrics of addod ATP in Extrrrctlrnt B scvcr~l times in other (unpublished) experiments shortly after substrate addition and it does appear to be a real c&ct. As yet. we cannot olfcr a satisfactory explanation, altll~~u~h it is being studied further in our I;rbor;ttory (MGa Di Nobili, personal communication). It does strongly suggest that soil ATP contents (corrected for individual recoveries of added ATP) measured in the glucose- or maize-amended soils, were greatly ovorcstimatcd up to day 5. When data obtained af day 5 were omitted, a very close linear relationship between biomass C and ATP w;ts obtained. irrespective of whsther the soils had rcceived glucose or maize [Fig. 3(a) and (b)]. If the soil ATP contents obtained at day 5 were corrected with the much higher percentage recoveries of ATP obtained at later sampling dates. the soil ATP contents measured at day 5 also fitted the regression well. This also suggested that these measured percentage recoveries of ATP were in error. between For glucose. the regression equation biomass C and soil ATP was: E, = (79.6 & 0.71) ATP. and. for maize: S, = (80.4 k0.88) ATP, both with high (r = 0.99) correlation cocficients {Fig. 3(a) and (b)]. Thus the biomass in both the low- and highmetal soils, with and without added substrates, had the same ATP concentration. The biomass ATP concentration meaned over both soils with and without added substrates (omitting the values obtained in amended soils at day 5) was 12.3 + 0.16pmol ATP g-’ biomass carbon. This value is close to that reported by Jenkinson (1988) of I I .7 ~trnol ATP g-’ biomass carbon, obtained from a large number of unamcnded soils taken from the literature and Ocio and Brookes (1990) for unamended soils and soils containing recently added wheat straw. This again suggests that ATP measurements provide a valid
following
substrate addition
More total and labelled CO, was evolved from the high-metal than the low-metal soil during the early, rapid decomposition phase following substrate addition. Once the tlush of decomposition was over (at 5 days for glucose and 50 days for maize). the mtes of CO: evolution declined to lower values than in the low-metal soil [Fig. 4(n) and (b)]. The amounts of total CO: evolved from the unamendcd low-metal soils wcrc l5-20% larger than the amounts cvolvcd from the high-rnet~l soil over the entire incubation period (data not given). In contrast, the specific respiration ratio [(biomass specific respiration rate in high-metal soil)/(spccific respiration rate in low-metal soil)] was about I.5 in the unamcndcd soils up to about day 25. After this time the difference in respiration rates became slightly less, so that between days 75 and 100 the specific respiration ratio was about 1.3 (Table 4). Following addition of glucose, the specific respiration rates were many times faster than in the unamcndcd soils betwccn days 0 and 5. Thereafter, they declined rapidly. However, the specific respiration ratios in the glucose-amended soils were similar at different sampling dates throughout the incubation. Thus, between days 0 and 25 the ratio was about 1.5, declining to about I.3 between days 25 and 50. This biomass specific respiration ratio between the glucose amended highand low-metal soils was therefore quite similar to the ratio between the unamended high- and low-metal soils. Biomass specific respiration rates in the maizeamended, high-metal soil were also consistently faster than in the maize-amended low-metal soil. Between days 0 and 25, the specific respiration ratio was about 2, declining to about 1.5 between days 25 and 100 (Table 4). These results therefore show that the smaller biomass in the high-metal soil (both substrate amended and unamended) had a faster specific respiration rate than the larger biomass in the low-metal soil. As a consequence of this, the ratio [(biomass lJC)/(respired “CO,)] was about 4@-50% less in the
Microbial
biomass dynamics in met&contaminated
8G (79.7
923
soils
f 0.7 1)ATP. 8
r * 0.99
EC*
35 ‘: 0,
(8’3.4!
0.89)ATP.
r * 0.99
84 .E n3 2 2
@D*.CYvG
;
i
x/@f:
0
i 50
100 BiomxK+
t50 carbon
200
250
305
(pg 9” soil)
The correlation between biomass C and soil ATP contents in the low- and high-metal soils. with and without addition of (a) “C-labellcd glucose and (b) “C4abelled tnaizc at difkrent sampling times. Note: The points 8. Q were from day 5 and were not included in the regression analysis (see text). Fig.
gfucosr-
3(a)
and
(b).
or maize-amen&d high-metal soifs during &composition phase (Q-5 days) (Tabfc 5). II is. however, probnbfy significant that the sum of biomass “C itnd rcspircd “CO, in the lowand high-metal soil Following addition of glucose (636 and 65Opg Cg _’ soil, rcspcctivefy) and maize (395 and 4% pg C g-l soil, r~spectjvely) were so cfose during the first 5 days following substrate addition. This the early
implies
in both the high- and abilities to metabolize the added gfucose-C or maize-C. However. much more of this C was converted t5 new biomass in the low- than in the high-metal soil. Conversely, much more of the available C fram the added substrate was evolved as C02-C from the biomass in the high-metaf than in the low-metal soil. low-met4
that
soil
the
biomass
had similar
Fig. J(a) and (h). Total and “C-lab&d CO: evolved from the low- and high-metal soils during incubation (4 “C4abclled glucose and (b) “C-labelled maize. The overall analytical coefficient of variation of all analyses is less than 5%.
K. CHASDER and P. C. BROOM TJblc
5. Biomass wds
“C.
dunng
“CO:
cvolvcd
the first 5 day
and the ratm
[(biomass
following
of incubation
“Cl (‘%IO: cvolvcdl~ III lowdddmon of “C-l.hclled glucox’ Sum
Btomass
“C
Amendment
LIlP c
GIUCOSC
Low-metal
soil
High-metal Maize
‘Amended bRatto The
with
sod
Low-merat
soil
High-metal
so11
&~ose
[(biomass
owall
“CO:
or maxe
“C,respired
coefficients
‘*CO:
P
of biomass
plus cwlwd
“C
“CO:
RJU0~
’ SOlI)
152
484
636
0.31
103
S?
650
0.19
I I4
181
395
0.40
70
336
JO6
0.21
as in Table
of varratxon
e\okd
Jnd hlgh.mctal or malzc’
2.
)I. for
biomass
“C
This increased diversion of C from biosynthesis to respired CO2 in the high-metal soil suggests that the metals are causing stress to the microbial biomass possibly by the mechanisms discussed in the Introduction. The stress is reflected in the higher specific respiration rates and consequently smaller amounts of substrate C incorporated into the microbial biomass in the high-metal soils. Recently, smaller biomasses were also found in metal-contaminated soils from other field experiments which rcccivcd past applications of metalcontaminated sewage-sludge. Chandcr and Brookcs (1991) reported that both Cu and Zn separately at 2-3 times current EC pcrmittcd limits decreased the amount of biomass by about 40% in both a sandyloam and silty-loam soil. However. the metal concentrations in thcsc soils were appreciably higher than at Wohurn. WC thorcforc suggest that our laboratory results may help to explain the previous finding of smaller b&masses in the high-metal soils of the Woburn Market Garden Experiment (Brookes and McGrath. 19x4). and thcso ctT’ccts may well operate at cvcn larger soil mctnl concentrations. However, extrapolation of results obtained under controlled laboratory conditions to those obtained from the field must always he done with caution. In particular, the biomass in the licld will receive small intermittent pulses of fresh substrates, such as roots, root exudates and plant rcsiducs. In contrast, in this work, both substrates were supplied as a single, large pulse, equivalent to 3.5 t C ha -’ in the O-23 cm plough layer. Also, while the mechanisms we have observed in this laboratory study probably also operate under field conditions, there is an ~tddition~tl possible mechanism. This is that the metals also exert direct toxic ctrects upon the plants thcmsolvcs, leading, in turn. to decreased inputs of plant-derived substrates and, consequently, to a smaller biomass. This possibility also requires investigation. Ac~~n,~l~,~f~r,r~t~,~~~.~ -K. Chandcr thanks the Commonwealth Commission and The British Council U.K. for financial support. We also thank R. Martens for supplying the “C-labellcd maize. S. P. McGrath for practical help and P. J. Harris, D. S. Jenkinson. S. P. McGrath and A. Wild for useful discussion.
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and
“CO,
evolved
are ‘.I
and
Z 6*..
reqxcwely.
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Microbtal
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dynamics
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in metal-contaminated
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925
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