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Mineralization of carbon from d - and l -amino acids and d -glucose in two contrasting soils
PII:
Soil Biol. Biochem. Vol. 30, No. 14, pp. 2009±2016, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(98)00075-3 0038-0717/98 $19.00 + 0.00
MINERALIZATION OF CARBON FROM D- AND L-AMINO ACIDS AND D-GLUCOSE IN TWO CONTRASTING SOILS R. W. O'DOWD and D. W. HOPKINS* Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, U.K. (Accepted 17 April 1998) SummaryÐFollowing addition of either the D- or the L-isomers of alanine, glutamine or glutamic acid or D-glucose, the CO2 production from an arable and a forest soil was measured until the pulses of CO2 production associated with substrate addition subsided. The maximum rate of additional CO2 production from the D-glucose amended soils occurred within the ®rst 48 h for both soils. The greatest rates of additional CO2 production from L-amino acid amended soils occurred within 108 h for the forest soil and 60 h for the arable soil. Following addition of D-amino acids to the forest soil, the maximum rate of additional CO2 production was less than that following addition of the corresponding Lamino acid addition. However, for this soil the pulse of additional CO2 production following D-amino acid amendment lasted longer and by the time it had subsided (360 h), the total additional CO2 production did not dier between isomeric forms of the same amino acid. Following D-amino acid addition to the arable soil, there were delays of between about 24 and 48 h before the onset of rapid additional CO2 production and the CO2 pulse subsided relatively rapidly. The total additional CO2 produced from the arable soil was signi®cantly less for the D-amino acid than for the corresponding L-amino acid treatments. Successive additions of D-glucose led to signi®cant increases in the subsequent rates of additional CO2 production from the forest soil, but not from the arable soil. Each successive L-amino acid amendment led to increases in the rate of additional CO2 production from both soils, as did successive additions of the D-amino acids to the forest soil. However, successive additions of the D-amino acids to the arable soil did not lead to consistent responses in the additional rate of CO2 production. # 1998 Elsevier Science Ltd. All rights reserved.
INTRODUCTION
The presence of an asymmetric carbon atom, i.e. one to which four dierent functional groups are attached, that acts as a chiral centre in the molecule means that all amino acids except glycine may exist as enantiomers (or D- and L-forms). Both isomeric forms of amino acids occur naturally, but in living organisms and their metabolites the L-isomers are far more abundant. The biological occurrence of Damino acids is restricted to a few amino acids with a few speci®c roles, such as the D-alanine and D-glutamic acid in the cross-linking of bacterial peptidoglycan (Weidel and Pelzer, 1964) and some antibiotics (Kuhn and Somerville, 1971). Amino acids are collectively an important component of the soil organic N pool (Stevenson, 1982) and they are likely, therefore, to be important natural sources of C and N for soil microorganisms. Most studies of amino acid biochemistry in soils have, quite correctly although implicitly, assumed that the L-amino acids are the predominant form. There have been very few studies of D-amino acid metabolism in soils. Waksman (1932) commented on chirality as a factor in¯uencing the rate of *Author for correspondence. E-mail: [email protected]
amino acid mineralization but, tantalisingly, did not say which isomeric form he thought was mineralized the more rapidly. It has also been suggested that bacterially-produced D-amino acids accumulate during the transformations of organic compounds in soil because of their persistence relative to that of the corresponding L-amino acids (Wagner and Mutatkar, 1968). This suggestion has been supported by little direct evidence, although D-alanine has been used as biomarker for bacteria (Gunnarson and Tunlid, 1986). Hopkins and O'Dowd (1997) have discussed chirality as a factor in¯uencing substrate utilization by soil microbial communities. Our previous studies have shown that short-term (usually 0±6 h) substrate induced respiratory responses following L-amino acid additions to a range of soils are greater than those following the addition of the corresponding D-amino acids (Hopkins and Ferguson, 1994; Hopkins et al., 1994, 1997; O'Dowd et al., 1997). Prior exposure of the soil microbial community to D-amino acids enhanced the subsequent rate of metabolism of the same D-amino acid to a proportionately greater extent than was observed for L-amino acids (Hopkins and Ferguson, 1994). Although the short-term L-amino acid induced respiration rate correlates well with mi-
2009
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R. W. O'Dowd and D. W. Hopkins
crobial biomass C, as determined by glucoseinduced respiration (Hopkins et al., 1994, 1997), Damino acid metabolism does not appear to be related to the activity of the bacterial component of the soil microbial community (O'Dowd et al., 1997), as we had earlier hypothesized (Hopkins and Ferguson, 1994). Using soils with a range of biological and chemical properties, Hopkins et al. (1997) showed that the ratio of the maximum shortterm rate of L-amino acid metabolism to that of the corresponding D-amino acid (the L-to-D ratio) declined markedly with both increasing activity per unit of biomass (CO2 production/biomass C; qCO2) and declining soil pH. This has led to the suggestion that increased L-to-D ratio is associated with increasing energetic demand by the soil community, so that the organisms are less fastidious with respect to C sources and will use relatively exotic C sources more readily in the presence of environmentally-imposed stress (Hopkins et al., 1997). This suggestion may be consistent with the recent observations of Staddon et al. (1997) that organic soils showed greater metabolic diversity than mineral soils, and has been supported indirectly by the observation that in soils with similar pH, the L-to-D ratio was much greater for soil with small available inorganic nutrient contents and organic C inputs compared to one with much larger available nutrient contents and C inputs (Hopkins and O'Dowd, 1997). To date our studies comparing D- and L-amino acid metabolism in soils have been restricted to short-term investigations in which only the immediate physiological response of the soil microbial community has been determined. The objectives of the work presented here were two-fold. First, to compare the patterns with time of CO2 production from soil following addition of D- and L-amino acids and D-glucose over a sucient period for the pulse of additional CO2 production to have subsided thereby allowing the substrate-induced CO2 production to be estimated. This will show whether the smaller amino acid induced respiration rates for D-amino acids compared to L-amino acids is due to a lag before the onset of C mineralization from Damino acids. Second, to determine how mineralization of C from the dierent isomeric forms of amino acids added to soils was aected by repeated prior exposure of the soil microbial community to the amino acid. Given that we have already shown
that prior exposure of the soil microbial community to an amino acid increases the subsequent substrate induced respiration rate to a greater extent for Damino acids than for L-amino acids, this second objective will show whether the prior exposure aects any lag period. MATERIALS AND METHODS
Soils Soils from the 0±15 cm depth at a forest and an arable site were collected, sieved (<4 mm) in the ®eld-moist state and stored at 48C for 2 weeks prior to experimentation. These soils were from the same sites as those used by O'Dowd et al. (1997) and they were selected because of their contrasting biological and physico±chemical characteristics (Table 1). Despite marked dierences in soil pH and organic matter content, both soils had similar microbial biomass C contents (D-glucose induced respiration), but the forest soil had a larger fungal component (ergosterol content). Carbon mineralization Two types of experiment were performed. These involved comparing the CO2 production from soils in response to either a single addition of substrate or in response to repeated substrate additions. In the ®rst experiment, 15 g soil (dry wt) at 50% water-holding capacity was amended with either the D- or L-isomer of alanine, glutamine or glutamic acid, or with D-glucose at the rate of 2 mg gÿ1 soil (dry wt). This amount of amino acid or glucose was sucient to induce the maximum short-term respiratory response in these soils (O'Dowd et al., 1997). The amino acids or glucose were mixed with 0.25 g talc as an inert carrier and then shaken with the soil to ensure thorough mixing. There were also controls comprising soils amended with talc only. Triplicate 2.5 g (dry wt) samples of each amended soil or of the corresponding unamended soils were weighed into glass vials and placed in miniaturised respirometric devices modi®ed from those of Heilmann and Beese (1992) as described by Hopkins and Ferguson (1994). CO2 accumulation in the headspace above the soil was determined at 24 or 48 h intervals by gas chromatography (thermal conductivity detector) as described by Hopkins and Ferguson (1994) until the pulse of CO2 production following amino acid or glucose amend-
Table 1. Details and properties of the soils (from O'Dowd et al., 1997) Soil Arable Forest
Organic mattera (%)
pH (water)
Biomass Cb (mg gÿ1 soil)
Ergosterol (mg gÿ1 soil)
6.1 22.4
6.8 3.9
0.42 (0.014) 0.53 (0.033)
0.16 (0.039) 4.38 (0.740)
Each value is the mean of 3 replicates and the standard deviation is shown in brackets. a % organic matter determined by loss on ignition. b Biomass C determined by glucose induced respiration method of Anderson and Domsch (1978).
D-
and L-amino acid mineralization
ment subsided. After each CO2 determination, the headspaces were ¯ushed with fresh air. The amounts of additional CO2 production resulting from amino acid or glucose addition to the soils were estimated from the dierence between CO2 from the amended and unamended soils. The basal respiration was estimated from the CO2 production from the unamended soil after the pulse of respiration at the start of the experiment had subsided. The second experiment was conducted in two phases with the CO2 production following repeated substrate addition to one soil being determined before that for the other. CO2 evolution from amended soils was determined as described above except that the soil samples used were collected at a dierent time from those in the ®rst experiment. When the pulse of CO2 production following the ®rst amino acid or glucose addition had subsided any moisture losses were restored by addition of distilled water and then, following a 7 d re-equilibration period, the soil was amended with a second and subsequently a third dose [both of 2 mg gÿ1 soil (dry wt)] of the respective amino acid or glucose. CO2 production following the second and third substrate amendments was determined as described above. The CO2 data from all experiments were expressed as the rate of CO2 production gÿ1 soil hÿ1 and analysed using two-way analysis of variance with substrate and time as the independent variables using the Minitab2 package (Ryan et al., 1984) for Microsoft Windows2. Four such analyses were carried out, one for each soil in the ®rst experiment and one for each soil in the second experiment. The statistical analyses were done in this way because within each type of experiment the sampling times varied between the soils. These dierences in sampling times arose because of the markedly dierent response times and rates of reaction between the soils. The signi®cance of dierences between the means for CO2 production within each of the four sub-sets of the data were tested using the least signi®cant dierences (at P < 0.05 and P < 0.01). The signi®cance of dierences between the means for CO2 production were between sub-sets of the data were tested using selected two-sample t-tests.
Fig. 1. Changes with time in the rates of CO2 production from the unamended soils (open symbols) and the rates of additional CO2 production following amendment with Dglucose (closed symbols). Each point is the mean of three replicates and the vertical bars represent the standard deviations
soils were similar (Table 1), the qCO2 for the forest soil was greater than that for the arable soil, and can be estimated as 77 and 50 nmol CO2 mgÿ1 biomass C hÿ1, respectively, using the biomass C values obtained from D-glucose induced respiration rates reported by O'Dowd et al. (1997) shown in Table 1. The greatest rate of additional CO2 production following addition of D-glucose occurred within the ®rst 48 h for both the arable and the forest soil (Fig. 1). The total additional amounts of CO2, above those from the unamended soils, produced following amendment with D-glucose corresponded to 20 and 28% of the C added as D-glucose for the forest and arable soils, respectively (Table 2). The greatest rates of additional CO2 production from L-amino acid amended soils occurred within 108 h for the forest soil (Fig. 2) and within 60 h for Table 2. Total additional CO2 production from forest and arable soils amended with D-glucose and the L- and D-isomers of amino acids Cumulative CO2 production (mmol CO2 gÿ1 soil)
RESULTS
The initial pulses of elevated CO2 evolution from the unamended soils subsided after 160 h in the forest soil and 48 h in the arable soil (Fig. 1). The individual rates of CO2 production following this time were used to estimate the basal respiration rates, which were 41 nmol CO2 gÿ1 soil hÿ1 (sd = 3.9; n = 5) and 21 nmol CO2 gÿ1 soil hÿ1 (sd = 9.6; n = 5) for the forest and the arable soils, respectively. Given that the biomass C contents of the two
2011
D-glucose L-alanine
D-alanine
L-glutamine
D-glutamine L-glutamic
D-glutamic
acid acid
forest soil
arable soil
13.4 21.5 21.8 19.6 20.0 21.6 19.4
19.0 29.0 17.2 27.7 13.8 27.1 15.1
(3.70) (3.91) (3.18) (2.49) (1.57) (2.05) (3.94)
(0.74) (5.09) (3.42) (1.31) (1.45) (1.36) (3.58)
The total periods over which CO2 evolution was measured were 360 h for the forest soil and 216 h for the arable soil. Each value is the mean of 3 replicates and the standard deviations are shown in brackets.
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R. W. O'Dowd and D. W. Hopkins
Fig. 2. Changes with time in the rates of additional CO2 production from the forest soil amended with the L-isomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine or glutamic acid. Each point is the mean of three replicates and the vertical bars represent the standard deviations. Note that the scales of the x-axes dier between Figs 2 and 3
the arable soil (Fig. 3). The maximum rates of additional CO2 production following L-amino acid addition were greater and subsided more rapidly for the arable than for the forest soil. The total amounts of CO2 evolved following L-amino acid amendment were signi®cantly greater for the arable than for the forest soil (Table 2). The additional CO2 production after amendment with D-amino acids to the forest soil followed a dierent pattern to that of the corresponding Lamino acids. For the D-amino acids, the rates of additional CO2 production increased more gradually and the periods of additional CO2 production lasted longer, but never exceeded the maximum rate for the corresponding L-amino acids (Fig. 2). Following D-amino acid addition to the forest soil, the maximum rate of additional CO2 production occurred at about 108 h, by which time it exceeded that from the corresponding L-amino acid-amended soils because CO2 production from the L-amino acidamended soils had started to subside by this time. By contrast with the D-amino acid amended forest soil, there were lags of between about 24 and 48 h before large increases in the rate of CO2 production from the arable soil amended with D-amino acids (Fig. 3). This was followed by a short pulse of CO2 production, which subsided relatively rapidly so that by about 60 h for D-glutamine and 132 h for both D-alanine and D-glutamic acid the rate of additional CO2 production was not signi®cantly dier-
ent to that from the corresponding L-amino acid amended soil (Fig. 3). The total additional amounts of CO2 released from the forest soil following amino acid amendment did not dier signi®cantly between either isomeric forms of the same amino acid or between dierent amino acids with the same con®guration and responded to a mean of 31% of the added C (Table 2). The total additional amounts of CO2 released from the arable soils following amino acid amendment were signi®cantly dierent between the dierent isomeric con®gurations, with a greater fraction of the added C having been lost from soil amended with the L-amino acids, but not between amino acids with the same isomeric con®guration (Table 2). The mean percentages of the added C lost as CO2 were 42% for the L-amino acids and 22% for the D-amino acids, respectively. Repeated additions of D-glucose led to signi®cant increases in the maximum rate at which additional CO2 was subsequently produced and in the total additional amount of C mineralized between the second and third glucose additions in the forest soil, but not in the arable soil (Fig. 4, Table 3). Repeated additions of L-alanine, L-glutamine and L-glutamic acid led to progressive increases in the rates of additional CO2 production from both soils (Figs 5 and 6), but the total additional CO2 produced only increased signi®cantly and consistently
Fig. 3. Changes with time in the rates of additional CO2 production from the arable soil amended with the L-isomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine and glutamic acid. Each point is the mean of three replicates and the vertical bars represent the standard deviations. Note that the scales of the x-axes dier between Figs 2 and 3
D-
and L-amino acid mineralization
2013
Fig. 4. Changes with time in the rates of additional CO2 production from the forest and the arable soil repeatedly amended with D-glucose. The times of each substrate addition are indicated by vertical arrows. Each point is the mean of three replicates and the vertical bars represent the standard deviations
for the forest soil (Table 3). In the arable soil, the total additional C mineralization with repeated Lamino acid additions increased signi®cantly between the ®rst and second L-amino acid additions, but not following the third addition (Table 3). In the case of L-glutamine, it declined signi®cantly after the third addition (Table 3). The eects of repeated additions of D-alanine, Dglutamine or D-glutamic acid to the forest soil were to reduce the period over which the additional CO2 was produced, in most cases to increase the maxi-
Fig. 5. Changes with time in the rates of additional CO2 production from the forest repeatedly amended with the Lisomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine and glutamic acid. The times of each substrate addition are indicated by vertical arrows. Each point is the mean of three replicates and the vertical bars represent the standard deviations
Table 3. Total additional CO2 production from the forest and arable soils following repeated amendment with D-glucose and the Land D-isomers of amino acids Cumulative CO2 production (mmol CO2 gÿ1 soil) addition D-glucose
L-alanine
D-alanine
L-glutamine
D-glutamine
L-glutamic
acid
D-glutamic
acid
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
forest soil
arable soil
14.0 13.9 22.6 27.1 30.1 36.9 23.1 27.5 35.5 25.2 31.1 40.1 24.1 31.7 29.2 27.2 29.6 36.1 24.2 23.9 28.4
16.4 17.1 15.3 25.6 33.8 30.2 20.1 29.2 24.8 28.4 38.7 31.1 20.2 35.7 15.5 22.5 34.9 33.6 14.6 32.3 23.2
(1.78) (2.59) (1.22) (1.82) (2.50) (1.07) (1.11) (0.91) (4.57) (2.97) (2.66) (0.43) (1.73) (5.75) (1.79) (2.85) (2.83) (1.63) (1.55) (1.80) (3.14)
(0.46) (0.97) (0.97) (1.54) (1.35) (1.60) (1.76) (1.21) (1.71) (0.69) (1.29) (0.80) (2.10) (1.41) (1.28) (3.89) (1.69) (2.23) (1.92) (1.85) (1.63)
Each value is the mean of 3 replicates and the standard deviations are shown in brackets.
Fig. 6. Changes with time in the rates of additional CO2 production from the arable soils amended with the L-isomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine and glutamic acid. The times of each substrate addition are indicated by vertical arrows. Each point is the mean of three replicates and the vertical bars represent the standard deviations
2014
R. W. O'Dowd and D. W. Hopkins
mum rate of CO2 production with subsequent additions (Fig. 5), and to increase the total additional C mineralized (Table 3). These increases were signi®cant with both the second and third additions of D-alanine, but in the case of D-glutamine, there was only a signi®cant increase between the ®rst and second additions. In the case of D-glutamic acid there was only a signi®cant increase between the ®rst and third additions (Table 3). In the arable soil, the eect of successive additions of D-amino acids was also to reduce the lag period before the onset of rapid CO2 production in all cases except following the third addition of glutamine (Fig. 6). By contrast with both the D- and Lamino acids additions to the forest soil and the Lamino acid additions to the arable soil, there were no consistent increases in the maximum rates of CO2 production with successive D-amino acid additions (Fig. 6). Also by contrast with the forest soil, the total additional C mineralized in the arable soil amended with D-amino acids increased signi®cantly between the ®rst and second amino acid additions but declined between the second and third additions (Table 3). This was similar to the eect of repeated L-amino acid additions to the arable soil. DISCUSSION
Dissolution of CO2 in the soil solution and its loss in the CO2/HCOÿ 3 equilibrium is a possible source of error in the arable soil, because of its neutral pH, which could have caused loss of CO2 from the head-space thereby leading to an apparent lag period. It seems, however, unlikely that this is a major source of error because although the bulk pH(water) was approximately 7.0, much of the soil solution would have been below pH 7.0. In addition, the buering capacity, which would also be positively related to CO2 loss from the head-space by this mechanism, is probably limited because of the relatively small clay and organic matter contents of this soil. Furthermore, the facts ®rst, that CO2 accumulated in the head-space above the D-glucoseamended arable soil within 24 h, which elicited a similar maximum CO2 production rate to the Damino acids, and second that the duration of the lag phase was apparently not inversely related to the maximum respiratory response from the Damino acids suggests that the lag periods observed for this soil with other substrates were genuine and not unduly in¯uenced by possible CO2 dissolution. Finally on this point, we have observed large L-to-D ratios (following short-term incubations) for soils between pH 5 and 6 with small qCO2 values, which is consistent with the slower rate of D-amino acid metabolism being due to a lag period. The patterns of CO2 evolution rate over time were similar for both soils when amended with any of the L-amino acids tested and with D-glucose,
being characterised by no detectable lag before the peak rate of CO2 production, followed by a rapid decline. A similar pattern of CO2 evolution following D-glucose addition to soil was reported by Tsai et al. (1997). These observations are consistent with our previous results in which short-term (0±6 h) respiration rates induced by dierent L-amino acids were well-correlated, both with each other and with those of D-glucose across a wide range of soils (Hopkins et al., 1994, 1997). Previously we have shown that the L-to-D ratios in short-term mineralization measurements were greater for soils with small qCO2 (Hopkins et al., 1997). The present data suggest that this is due to the short-term assays coinciding with a lag period before the onset of rapid D-amino acid induced CO2 production in low qCO2 soils, such as the arable soil, whereas there was no such lag in high qCO2 soils, such as the forest soil. The presence of a lag period in the arable soil indicates that the ability to metabolise D-amino acids was being initiated (by induction or de-repression) in all or some of the microorganisms (i.e. a physiological response), or that a change in community structure was occurring in favour of organisms capable of Damino acid metabolism (i.e. a selective growth response), or that both were occurring. Both these responses are consistent with the reductions in the duration of the lag periods with successive D-amino acid addition to the arable soil and the enhancements in the rates of D-amino acid mineralization on repeated exposure observed here and previously (Hopkins and Ferguson, 1994). By contrast with the arable soil, rapid metabolism of D-amino acids occurred in the forest soil from the outset. Since the forest soil had a much larger fungal content, this observation adds further weight to the conclusion of O'Dowd et al. (1997) that D-amino acid metabolism is not solely a function of bacterial activity, even though bacteria are probably the main biological source of D-amino acids in soils. More important, however, is the question of why should the microbial community in the forest soil, which neither has a large bacterial content nor receives signi®cant inputs of faecal material that may be enriched in bacterially-produced compounds, possess the ability to metabolise Damino acids rapidly from the outset? We have previously suggested that this is due to soil microbial communities being less fastidious under conditions where they have a high energetic demand as indicated by high qCO2 (Hopkins et al., 1997; Hopkins and O'Dowd, 1997). The present results suggest that by comparison with that of the arable soil, the microbial community in the forest soil was under greater physiological stress, consistent with its greater qCO2, which could be related in part to low soil pH or other factors such as poorer resource quality in the forest soil.
D-
and L-amino acid mineralization
The fact that the amount of CO2 lost from the forest soil did not dier with the isomeric form of the amino acid added suggests that the rate of metabolism was the main dierence between isomers in the forest soil. It is possible that D-amino acid utilization by soil microorganisms occurred via a D-enantiospeci®c pathway, alternatively, the slower mineralization rate may have been due to a speci®c metabolic step, such as D-amino acid deamination (which would yield an achiral keto-acid) or racemisation, being required for D-amino acid metabolism compared with L-amino acids in the forest soil. Tebbe and Reber (1991) suggest that the ®rst stage in the degradation of phosphinothricin, a D-amino acid analogue, is racemisation but we cannot be sure that racemisation is the ®rst stage in the breakdown of D-amino acids in the forest soil. However, irrespective of whether the ®rst step in Damino acid metabolism in the forest soil is racemisation or deamination, it is possible that after this initial step the routes of metabolism of the dierent enantiomeric forms of amino acids were similar in this soil. In the forest soil both the maximum rate and the amount of CO2 released increased with each successive addition of substrate, whether the substrate was D-glucose, an L-amino acid or a D-amino acid. This suggests that the availability of readily-metabolisable C was the main constraint on microbial activity, and that there were neither strong preferences for the biochemical form nor for the enantiomeric con®guration of the organic molecules supplied. This is consistent with the observation of a lack of enantioselective preference by nutritionally-stressed soil microorganisms (Hopkins and O'Dowd, 1997). By contrast, in the arable soil the most marked increases in maximum rate of CO2 production and the total amount of CO2 released with successive substrate additions occurred for the amino acids and not for D-glucose. This suggests that in the arable soil, the availability of N rather than organic C may have been an important constraint on microbial activity, but and that even under such a constraint, D-amino acids were not as readily metabolised as L-amino acids. It is possible, therefore, that the ability of soil microorganisms to utilise D-amino acids is more closely related to their demand for C than for N. If this is the case, deamination could be the ®rst step in D-amino acid utilization in the arable soil. In this study we have extended our observations on the comparison of D- and L-amino acid metabolism in soil from short- to longer-term mineralization measurements. These observations have con®rmed the expected similarity between the patterns of CO2 mineralization from D-glucose and Lamino acids in contrasting soils, but have also demonstrated that important dierences in mineralization of dierent isomeric forms of amino acids
2015
occur between soils. First, our previous observation that the short-term rate of rapid D-amino acid induced respiration was not a constant fraction of that of the corresponding L-amino acid is apparently related to a lag before the onset of rapid Damino acid metabolism in soils with large L-to-D ratios. Second, even after the lag phase, the total C mineralized to CO2 from D-amino acids was less than that observed in a soil for which there was no initial lag. This suggests dierent routes of D-amino acid metabolism or dierent eciencies of utilization of D-amino acids as substrates between soils. Third, in soil for which there was no lag before the onset of rapid D-amino acid mineralization, some other factor, possibly deamination or racemisation, limited the maximum rate of mineralization compared with that of the corresponding L-amino acid. AcknowledgementsÐWe are grateful to the Department of Agriculture for Northern Ireland and the U.K. Biotechnology and Biological Sciences Research Council (Soil-Plant-Microbe Interactions initiative) for their respective contributions to a postgraduate studentship for RWO.
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Anderson J. P. E. and Domsch K. H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215±221. Gunnarson T. and Tunlid A. (1986) Recycling of fecal pellets in isopods: microorganisms and nitrogen compounds as potential food for Oniscus asellus L.. Soil Biology & Biochemistry 18, 595±600. Heilmann B. and Beese F. (1992) Miniaturized method to measure carbon dioxide production and biomass of soil microorganisms. Soil Science Society of America Journal 56, 596±598. Hopkins D. W. and Ferguson K. E. (1994) Substrate induced respiration in soil amended with dierent amino acid isomers. Applied Soil Ecology 1, 75±81. Hopkins D. W. and O'Dowd R. W. (1997) Chirality is a factor in substrate utilization. In Microbial Communities: Structural vs Functional Approaches, ed H. Insam and A. Rangger, pp. 215±228. Springer-Verlag, Berlin. Hopkins D. W., Isabella B. L. and Scott S. E. (1994) Relationship between microbial biomass and substrate induced respiration in soil amended with D- and L-isomers of amino acids. Soil Biology & Biochemistry 26, 1623±1627. Hopkin D. W., O'Dowd R. W. and Shiel R. S. (1997) Comparison of D- and L-amino acid metabolism in soils with diering microbial biomass and activity. Soil Biology & Biochemistry 29, 23±29. Kuhn J. and Somerville R. (1971) Mutant strains of Escherichia coli that use D-amino acids. Proceedings of the National Academy of Sciences (Washington), Vol. 68, pp. 2484±2487. O'Dowd R. W., Parsons R. and Hopkins D. W. (1997) Soil respiration induced by the D- and L- isomers of a range of amino acids. Soil Biology & Biochemistry 29, 1665±1671. Ryan B. F., Joiner B. L. and Ryan T. A. (1984) Minitab Handbook. Duxbury Press, Boston.
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tory conditions: Eects of concentration and soil amendments on 14 CO2 production. Biology and Fertility of Soils 11, 62±67. Wagner G. H. and Mutatkar V. K. (1968) Amino acid components of soil organic matter formed during humi®cation of 14 C from glucose. Soil Science Society of America Proceedings 32, 683±684. Waksman S. A. (1932) Principles of Soil Microbiology, 2nd ed. BaillieÁre, Tindall and Cox, London. Weidel W. and Pelzer H. (1964) Bag-shaped macromolecules Ð a new outlook on bacterial cell walls. Advances in Enzymology 26, 193±232.
Soil Biol. Biochem. Vol. 30, No. 14, pp. 2009±2016, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(98)00075-3 0038-0717/98 $19.00 + 0.00
MINERALIZATION OF CARBON FROM D- AND L-AMINO ACIDS AND D-GLUCOSE IN TWO CONTRASTING SOILS R. W. O'DOWD and D. W. HOPKINS* Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, U.K. (Accepted 17 April 1998) SummaryÐFollowing addition of either the D- or the L-isomers of alanine, glutamine or glutamic acid or D-glucose, the CO2 production from an arable and a forest soil was measured until the pulses of CO2 production associated with substrate addition subsided. The maximum rate of additional CO2 production from the D-glucose amended soils occurred within the ®rst 48 h for both soils. The greatest rates of additional CO2 production from L-amino acid amended soils occurred within 108 h for the forest soil and 60 h for the arable soil. Following addition of D-amino acids to the forest soil, the maximum rate of additional CO2 production was less than that following addition of the corresponding Lamino acid addition. However, for this soil the pulse of additional CO2 production following D-amino acid amendment lasted longer and by the time it had subsided (360 h), the total additional CO2 production did not dier between isomeric forms of the same amino acid. Following D-amino acid addition to the arable soil, there were delays of between about 24 and 48 h before the onset of rapid additional CO2 production and the CO2 pulse subsided relatively rapidly. The total additional CO2 produced from the arable soil was signi®cantly less for the D-amino acid than for the corresponding L-amino acid treatments. Successive additions of D-glucose led to signi®cant increases in the subsequent rates of additional CO2 production from the forest soil, but not from the arable soil. Each successive L-amino acid amendment led to increases in the rate of additional CO2 production from both soils, as did successive additions of the D-amino acids to the forest soil. However, successive additions of the D-amino acids to the arable soil did not lead to consistent responses in the additional rate of CO2 production. # 1998 Elsevier Science Ltd. All rights reserved.
INTRODUCTION
The presence of an asymmetric carbon atom, i.e. one to which four dierent functional groups are attached, that acts as a chiral centre in the molecule means that all amino acids except glycine may exist as enantiomers (or D- and L-forms). Both isomeric forms of amino acids occur naturally, but in living organisms and their metabolites the L-isomers are far more abundant. The biological occurrence of Damino acids is restricted to a few amino acids with a few speci®c roles, such as the D-alanine and D-glutamic acid in the cross-linking of bacterial peptidoglycan (Weidel and Pelzer, 1964) and some antibiotics (Kuhn and Somerville, 1971). Amino acids are collectively an important component of the soil organic N pool (Stevenson, 1982) and they are likely, therefore, to be important natural sources of C and N for soil microorganisms. Most studies of amino acid biochemistry in soils have, quite correctly although implicitly, assumed that the L-amino acids are the predominant form. There have been very few studies of D-amino acid metabolism in soils. Waksman (1932) commented on chirality as a factor in¯uencing the rate of *Author for correspondence. E-mail: [email protected]
amino acid mineralization but, tantalisingly, did not say which isomeric form he thought was mineralized the more rapidly. It has also been suggested that bacterially-produced D-amino acids accumulate during the transformations of organic compounds in soil because of their persistence relative to that of the corresponding L-amino acids (Wagner and Mutatkar, 1968). This suggestion has been supported by little direct evidence, although D-alanine has been used as biomarker for bacteria (Gunnarson and Tunlid, 1986). Hopkins and O'Dowd (1997) have discussed chirality as a factor in¯uencing substrate utilization by soil microbial communities. Our previous studies have shown that short-term (usually 0±6 h) substrate induced respiratory responses following L-amino acid additions to a range of soils are greater than those following the addition of the corresponding D-amino acids (Hopkins and Ferguson, 1994; Hopkins et al., 1994, 1997; O'Dowd et al., 1997). Prior exposure of the soil microbial community to D-amino acids enhanced the subsequent rate of metabolism of the same D-amino acid to a proportionately greater extent than was observed for L-amino acids (Hopkins and Ferguson, 1994). Although the short-term L-amino acid induced respiration rate correlates well with mi-
2009
2010
R. W. O'Dowd and D. W. Hopkins
crobial biomass C, as determined by glucoseinduced respiration (Hopkins et al., 1994, 1997), Damino acid metabolism does not appear to be related to the activity of the bacterial component of the soil microbial community (O'Dowd et al., 1997), as we had earlier hypothesized (Hopkins and Ferguson, 1994). Using soils with a range of biological and chemical properties, Hopkins et al. (1997) showed that the ratio of the maximum shortterm rate of L-amino acid metabolism to that of the corresponding D-amino acid (the L-to-D ratio) declined markedly with both increasing activity per unit of biomass (CO2 production/biomass C; qCO2) and declining soil pH. This has led to the suggestion that increased L-to-D ratio is associated with increasing energetic demand by the soil community, so that the organisms are less fastidious with respect to C sources and will use relatively exotic C sources more readily in the presence of environmentally-imposed stress (Hopkins et al., 1997). This suggestion may be consistent with the recent observations of Staddon et al. (1997) that organic soils showed greater metabolic diversity than mineral soils, and has been supported indirectly by the observation that in soils with similar pH, the L-to-D ratio was much greater for soil with small available inorganic nutrient contents and organic C inputs compared to one with much larger available nutrient contents and C inputs (Hopkins and O'Dowd, 1997). To date our studies comparing D- and L-amino acid metabolism in soils have been restricted to short-term investigations in which only the immediate physiological response of the soil microbial community has been determined. The objectives of the work presented here were two-fold. First, to compare the patterns with time of CO2 production from soil following addition of D- and L-amino acids and D-glucose over a sucient period for the pulse of additional CO2 production to have subsided thereby allowing the substrate-induced CO2 production to be estimated. This will show whether the smaller amino acid induced respiration rates for D-amino acids compared to L-amino acids is due to a lag before the onset of C mineralization from Damino acids. Second, to determine how mineralization of C from the dierent isomeric forms of amino acids added to soils was aected by repeated prior exposure of the soil microbial community to the amino acid. Given that we have already shown
that prior exposure of the soil microbial community to an amino acid increases the subsequent substrate induced respiration rate to a greater extent for Damino acids than for L-amino acids, this second objective will show whether the prior exposure aects any lag period. MATERIALS AND METHODS
Soils Soils from the 0±15 cm depth at a forest and an arable site were collected, sieved (<4 mm) in the ®eld-moist state and stored at 48C for 2 weeks prior to experimentation. These soils were from the same sites as those used by O'Dowd et al. (1997) and they were selected because of their contrasting biological and physico±chemical characteristics (Table 1). Despite marked dierences in soil pH and organic matter content, both soils had similar microbial biomass C contents (D-glucose induced respiration), but the forest soil had a larger fungal component (ergosterol content). Carbon mineralization Two types of experiment were performed. These involved comparing the CO2 production from soils in response to either a single addition of substrate or in response to repeated substrate additions. In the ®rst experiment, 15 g soil (dry wt) at 50% water-holding capacity was amended with either the D- or L-isomer of alanine, glutamine or glutamic acid, or with D-glucose at the rate of 2 mg gÿ1 soil (dry wt). This amount of amino acid or glucose was sucient to induce the maximum short-term respiratory response in these soils (O'Dowd et al., 1997). The amino acids or glucose were mixed with 0.25 g talc as an inert carrier and then shaken with the soil to ensure thorough mixing. There were also controls comprising soils amended with talc only. Triplicate 2.5 g (dry wt) samples of each amended soil or of the corresponding unamended soils were weighed into glass vials and placed in miniaturised respirometric devices modi®ed from those of Heilmann and Beese (1992) as described by Hopkins and Ferguson (1994). CO2 accumulation in the headspace above the soil was determined at 24 or 48 h intervals by gas chromatography (thermal conductivity detector) as described by Hopkins and Ferguson (1994) until the pulse of CO2 production following amino acid or glucose amend-
Table 1. Details and properties of the soils (from O'Dowd et al., 1997) Soil Arable Forest
Organic mattera (%)
pH (water)
Biomass Cb (mg gÿ1 soil)
Ergosterol (mg gÿ1 soil)
6.1 22.4
6.8 3.9
0.42 (0.014) 0.53 (0.033)
0.16 (0.039) 4.38 (0.740)
Each value is the mean of 3 replicates and the standard deviation is shown in brackets. a % organic matter determined by loss on ignition. b Biomass C determined by glucose induced respiration method of Anderson and Domsch (1978).
D-
and L-amino acid mineralization
ment subsided. After each CO2 determination, the headspaces were ¯ushed with fresh air. The amounts of additional CO2 production resulting from amino acid or glucose addition to the soils were estimated from the dierence between CO2 from the amended and unamended soils. The basal respiration was estimated from the CO2 production from the unamended soil after the pulse of respiration at the start of the experiment had subsided. The second experiment was conducted in two phases with the CO2 production following repeated substrate addition to one soil being determined before that for the other. CO2 evolution from amended soils was determined as described above except that the soil samples used were collected at a dierent time from those in the ®rst experiment. When the pulse of CO2 production following the ®rst amino acid or glucose addition had subsided any moisture losses were restored by addition of distilled water and then, following a 7 d re-equilibration period, the soil was amended with a second and subsequently a third dose [both of 2 mg gÿ1 soil (dry wt)] of the respective amino acid or glucose. CO2 production following the second and third substrate amendments was determined as described above. The CO2 data from all experiments were expressed as the rate of CO2 production gÿ1 soil hÿ1 and analysed using two-way analysis of variance with substrate and time as the independent variables using the Minitab2 package (Ryan et al., 1984) for Microsoft Windows2. Four such analyses were carried out, one for each soil in the ®rst experiment and one for each soil in the second experiment. The statistical analyses were done in this way because within each type of experiment the sampling times varied between the soils. These dierences in sampling times arose because of the markedly dierent response times and rates of reaction between the soils. The signi®cance of dierences between the means for CO2 production within each of the four sub-sets of the data were tested using the least signi®cant dierences (at P < 0.05 and P < 0.01). The signi®cance of dierences between the means for CO2 production were between sub-sets of the data were tested using selected two-sample t-tests.
Fig. 1. Changes with time in the rates of CO2 production from the unamended soils (open symbols) and the rates of additional CO2 production following amendment with Dglucose (closed symbols). Each point is the mean of three replicates and the vertical bars represent the standard deviations
soils were similar (Table 1), the qCO2 for the forest soil was greater than that for the arable soil, and can be estimated as 77 and 50 nmol CO2 mgÿ1 biomass C hÿ1, respectively, using the biomass C values obtained from D-glucose induced respiration rates reported by O'Dowd et al. (1997) shown in Table 1. The greatest rate of additional CO2 production following addition of D-glucose occurred within the ®rst 48 h for both the arable and the forest soil (Fig. 1). The total additional amounts of CO2, above those from the unamended soils, produced following amendment with D-glucose corresponded to 20 and 28% of the C added as D-glucose for the forest and arable soils, respectively (Table 2). The greatest rates of additional CO2 production from L-amino acid amended soils occurred within 108 h for the forest soil (Fig. 2) and within 60 h for Table 2. Total additional CO2 production from forest and arable soils amended with D-glucose and the L- and D-isomers of amino acids Cumulative CO2 production (mmol CO2 gÿ1 soil)
RESULTS
The initial pulses of elevated CO2 evolution from the unamended soils subsided after 160 h in the forest soil and 48 h in the arable soil (Fig. 1). The individual rates of CO2 production following this time were used to estimate the basal respiration rates, which were 41 nmol CO2 gÿ1 soil hÿ1 (sd = 3.9; n = 5) and 21 nmol CO2 gÿ1 soil hÿ1 (sd = 9.6; n = 5) for the forest and the arable soils, respectively. Given that the biomass C contents of the two
2011
D-glucose L-alanine
D-alanine
L-glutamine
D-glutamine L-glutamic
D-glutamic
acid acid
forest soil
arable soil
13.4 21.5 21.8 19.6 20.0 21.6 19.4
19.0 29.0 17.2 27.7 13.8 27.1 15.1
(3.70) (3.91) (3.18) (2.49) (1.57) (2.05) (3.94)
(0.74) (5.09) (3.42) (1.31) (1.45) (1.36) (3.58)
The total periods over which CO2 evolution was measured were 360 h for the forest soil and 216 h for the arable soil. Each value is the mean of 3 replicates and the standard deviations are shown in brackets.
2012
R. W. O'Dowd and D. W. Hopkins
Fig. 2. Changes with time in the rates of additional CO2 production from the forest soil amended with the L-isomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine or glutamic acid. Each point is the mean of three replicates and the vertical bars represent the standard deviations. Note that the scales of the x-axes dier between Figs 2 and 3
the arable soil (Fig. 3). The maximum rates of additional CO2 production following L-amino acid addition were greater and subsided more rapidly for the arable than for the forest soil. The total amounts of CO2 evolved following L-amino acid amendment were signi®cantly greater for the arable than for the forest soil (Table 2). The additional CO2 production after amendment with D-amino acids to the forest soil followed a dierent pattern to that of the corresponding Lamino acids. For the D-amino acids, the rates of additional CO2 production increased more gradually and the periods of additional CO2 production lasted longer, but never exceeded the maximum rate for the corresponding L-amino acids (Fig. 2). Following D-amino acid addition to the forest soil, the maximum rate of additional CO2 production occurred at about 108 h, by which time it exceeded that from the corresponding L-amino acid-amended soils because CO2 production from the L-amino acidamended soils had started to subside by this time. By contrast with the D-amino acid amended forest soil, there were lags of between about 24 and 48 h before large increases in the rate of CO2 production from the arable soil amended with D-amino acids (Fig. 3). This was followed by a short pulse of CO2 production, which subsided relatively rapidly so that by about 60 h for D-glutamine and 132 h for both D-alanine and D-glutamic acid the rate of additional CO2 production was not signi®cantly dier-
ent to that from the corresponding L-amino acid amended soil (Fig. 3). The total additional amounts of CO2 released from the forest soil following amino acid amendment did not dier signi®cantly between either isomeric forms of the same amino acid or between dierent amino acids with the same con®guration and responded to a mean of 31% of the added C (Table 2). The total additional amounts of CO2 released from the arable soils following amino acid amendment were signi®cantly dierent between the dierent isomeric con®gurations, with a greater fraction of the added C having been lost from soil amended with the L-amino acids, but not between amino acids with the same isomeric con®guration (Table 2). The mean percentages of the added C lost as CO2 were 42% for the L-amino acids and 22% for the D-amino acids, respectively. Repeated additions of D-glucose led to signi®cant increases in the maximum rate at which additional CO2 was subsequently produced and in the total additional amount of C mineralized between the second and third glucose additions in the forest soil, but not in the arable soil (Fig. 4, Table 3). Repeated additions of L-alanine, L-glutamine and L-glutamic acid led to progressive increases in the rates of additional CO2 production from both soils (Figs 5 and 6), but the total additional CO2 produced only increased signi®cantly and consistently
Fig. 3. Changes with time in the rates of additional CO2 production from the arable soil amended with the L-isomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine and glutamic acid. Each point is the mean of three replicates and the vertical bars represent the standard deviations. Note that the scales of the x-axes dier between Figs 2 and 3
D-
and L-amino acid mineralization
2013
Fig. 4. Changes with time in the rates of additional CO2 production from the forest and the arable soil repeatedly amended with D-glucose. The times of each substrate addition are indicated by vertical arrows. Each point is the mean of three replicates and the vertical bars represent the standard deviations
for the forest soil (Table 3). In the arable soil, the total additional C mineralization with repeated Lamino acid additions increased signi®cantly between the ®rst and second L-amino acid additions, but not following the third addition (Table 3). In the case of L-glutamine, it declined signi®cantly after the third addition (Table 3). The eects of repeated additions of D-alanine, Dglutamine or D-glutamic acid to the forest soil were to reduce the period over which the additional CO2 was produced, in most cases to increase the maxi-
Fig. 5. Changes with time in the rates of additional CO2 production from the forest repeatedly amended with the Lisomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine and glutamic acid. The times of each substrate addition are indicated by vertical arrows. Each point is the mean of three replicates and the vertical bars represent the standard deviations
Table 3. Total additional CO2 production from the forest and arable soils following repeated amendment with D-glucose and the Land D-isomers of amino acids Cumulative CO2 production (mmol CO2 gÿ1 soil) addition D-glucose
L-alanine
D-alanine
L-glutamine
D-glutamine
L-glutamic
acid
D-glutamic
acid
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
forest soil
arable soil
14.0 13.9 22.6 27.1 30.1 36.9 23.1 27.5 35.5 25.2 31.1 40.1 24.1 31.7 29.2 27.2 29.6 36.1 24.2 23.9 28.4
16.4 17.1 15.3 25.6 33.8 30.2 20.1 29.2 24.8 28.4 38.7 31.1 20.2 35.7 15.5 22.5 34.9 33.6 14.6 32.3 23.2
(1.78) (2.59) (1.22) (1.82) (2.50) (1.07) (1.11) (0.91) (4.57) (2.97) (2.66) (0.43) (1.73) (5.75) (1.79) (2.85) (2.83) (1.63) (1.55) (1.80) (3.14)
(0.46) (0.97) (0.97) (1.54) (1.35) (1.60) (1.76) (1.21) (1.71) (0.69) (1.29) (0.80) (2.10) (1.41) (1.28) (3.89) (1.69) (2.23) (1.92) (1.85) (1.63)
Each value is the mean of 3 replicates and the standard deviations are shown in brackets.
Fig. 6. Changes with time in the rates of additional CO2 production from the arable soils amended with the L-isomers (open symbols) and the D-isomers (closed symbols) of alanine, glutamine and glutamic acid. The times of each substrate addition are indicated by vertical arrows. Each point is the mean of three replicates and the vertical bars represent the standard deviations
2014
R. W. O'Dowd and D. W. Hopkins
mum rate of CO2 production with subsequent additions (Fig. 5), and to increase the total additional C mineralized (Table 3). These increases were signi®cant with both the second and third additions of D-alanine, but in the case of D-glutamine, there was only a signi®cant increase between the ®rst and second additions. In the case of D-glutamic acid there was only a signi®cant increase between the ®rst and third additions (Table 3). In the arable soil, the eect of successive additions of D-amino acids was also to reduce the lag period before the onset of rapid CO2 production in all cases except following the third addition of glutamine (Fig. 6). By contrast with both the D- and Lamino acids additions to the forest soil and the Lamino acid additions to the arable soil, there were no consistent increases in the maximum rates of CO2 production with successive D-amino acid additions (Fig. 6). Also by contrast with the forest soil, the total additional C mineralized in the arable soil amended with D-amino acids increased signi®cantly between the ®rst and second amino acid additions but declined between the second and third additions (Table 3). This was similar to the eect of repeated L-amino acid additions to the arable soil. DISCUSSION
Dissolution of CO2 in the soil solution and its loss in the CO2/HCOÿ 3 equilibrium is a possible source of error in the arable soil, because of its neutral pH, which could have caused loss of CO2 from the head-space thereby leading to an apparent lag period. It seems, however, unlikely that this is a major source of error because although the bulk pH(water) was approximately 7.0, much of the soil solution would have been below pH 7.0. In addition, the buering capacity, which would also be positively related to CO2 loss from the head-space by this mechanism, is probably limited because of the relatively small clay and organic matter contents of this soil. Furthermore, the facts ®rst, that CO2 accumulated in the head-space above the D-glucoseamended arable soil within 24 h, which elicited a similar maximum CO2 production rate to the Damino acids, and second that the duration of the lag phase was apparently not inversely related to the maximum respiratory response from the Damino acids suggests that the lag periods observed for this soil with other substrates were genuine and not unduly in¯uenced by possible CO2 dissolution. Finally on this point, we have observed large L-to-D ratios (following short-term incubations) for soils between pH 5 and 6 with small qCO2 values, which is consistent with the slower rate of D-amino acid metabolism being due to a lag period. The patterns of CO2 evolution rate over time were similar for both soils when amended with any of the L-amino acids tested and with D-glucose,
being characterised by no detectable lag before the peak rate of CO2 production, followed by a rapid decline. A similar pattern of CO2 evolution following D-glucose addition to soil was reported by Tsai et al. (1997). These observations are consistent with our previous results in which short-term (0±6 h) respiration rates induced by dierent L-amino acids were well-correlated, both with each other and with those of D-glucose across a wide range of soils (Hopkins et al., 1994, 1997). Previously we have shown that the L-to-D ratios in short-term mineralization measurements were greater for soils with small qCO2 (Hopkins et al., 1997). The present data suggest that this is due to the short-term assays coinciding with a lag period before the onset of rapid D-amino acid induced CO2 production in low qCO2 soils, such as the arable soil, whereas there was no such lag in high qCO2 soils, such as the forest soil. The presence of a lag period in the arable soil indicates that the ability to metabolise D-amino acids was being initiated (by induction or de-repression) in all or some of the microorganisms (i.e. a physiological response), or that a change in community structure was occurring in favour of organisms capable of Damino acid metabolism (i.e. a selective growth response), or that both were occurring. Both these responses are consistent with the reductions in the duration of the lag periods with successive D-amino acid addition to the arable soil and the enhancements in the rates of D-amino acid mineralization on repeated exposure observed here and previously (Hopkins and Ferguson, 1994). By contrast with the arable soil, rapid metabolism of D-amino acids occurred in the forest soil from the outset. Since the forest soil had a much larger fungal content, this observation adds further weight to the conclusion of O'Dowd et al. (1997) that D-amino acid metabolism is not solely a function of bacterial activity, even though bacteria are probably the main biological source of D-amino acids in soils. More important, however, is the question of why should the microbial community in the forest soil, which neither has a large bacterial content nor receives signi®cant inputs of faecal material that may be enriched in bacterially-produced compounds, possess the ability to metabolise Damino acids rapidly from the outset? We have previously suggested that this is due to soil microbial communities being less fastidious under conditions where they have a high energetic demand as indicated by high qCO2 (Hopkins et al., 1997; Hopkins and O'Dowd, 1997). The present results suggest that by comparison with that of the arable soil, the microbial community in the forest soil was under greater physiological stress, consistent with its greater qCO2, which could be related in part to low soil pH or other factors such as poorer resource quality in the forest soil.
D-
and L-amino acid mineralization
The fact that the amount of CO2 lost from the forest soil did not dier with the isomeric form of the amino acid added suggests that the rate of metabolism was the main dierence between isomers in the forest soil. It is possible that D-amino acid utilization by soil microorganisms occurred via a D-enantiospeci®c pathway, alternatively, the slower mineralization rate may have been due to a speci®c metabolic step, such as D-amino acid deamination (which would yield an achiral keto-acid) or racemisation, being required for D-amino acid metabolism compared with L-amino acids in the forest soil. Tebbe and Reber (1991) suggest that the ®rst stage in the degradation of phosphinothricin, a D-amino acid analogue, is racemisation but we cannot be sure that racemisation is the ®rst stage in the breakdown of D-amino acids in the forest soil. However, irrespective of whether the ®rst step in Damino acid metabolism in the forest soil is racemisation or deamination, it is possible that after this initial step the routes of metabolism of the dierent enantiomeric forms of amino acids were similar in this soil. In the forest soil both the maximum rate and the amount of CO2 released increased with each successive addition of substrate, whether the substrate was D-glucose, an L-amino acid or a D-amino acid. This suggests that the availability of readily-metabolisable C was the main constraint on microbial activity, and that there were neither strong preferences for the biochemical form nor for the enantiomeric con®guration of the organic molecules supplied. This is consistent with the observation of a lack of enantioselective preference by nutritionally-stressed soil microorganisms (Hopkins and O'Dowd, 1997). By contrast, in the arable soil the most marked increases in maximum rate of CO2 production and the total amount of CO2 released with successive substrate additions occurred for the amino acids and not for D-glucose. This suggests that in the arable soil, the availability of N rather than organic C may have been an important constraint on microbial activity, but and that even under such a constraint, D-amino acids were not as readily metabolised as L-amino acids. It is possible, therefore, that the ability of soil microorganisms to utilise D-amino acids is more closely related to their demand for C than for N. If this is the case, deamination could be the ®rst step in D-amino acid utilization in the arable soil. In this study we have extended our observations on the comparison of D- and L-amino acid metabolism in soil from short- to longer-term mineralization measurements. These observations have con®rmed the expected similarity between the patterns of CO2 mineralization from D-glucose and Lamino acids in contrasting soils, but have also demonstrated that important dierences in mineralization of dierent isomeric forms of amino acids
2015
occur between soils. First, our previous observation that the short-term rate of rapid D-amino acid induced respiration was not a constant fraction of that of the corresponding L-amino acid is apparently related to a lag before the onset of rapid Damino acid metabolism in soils with large L-to-D ratios. Second, even after the lag phase, the total C mineralized to CO2 from D-amino acids was less than that observed in a soil for which there was no initial lag. This suggests dierent routes of D-amino acid metabolism or dierent eciencies of utilization of D-amino acids as substrates between soils. Third, in soil for which there was no lag before the onset of rapid D-amino acid mineralization, some other factor, possibly deamination or racemisation, limited the maximum rate of mineralization compared with that of the corresponding L-amino acid. AcknowledgementsÐWe are grateful to the Department of Agriculture for Northern Ireland and the U.K. Biotechnology and Biological Sciences Research Council (Soil-Plant-Microbe Interactions initiative) for their respective contributions to a postgraduate studentship for RWO.
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Anderson J. P. E. and Domsch K. H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215±221. Gunnarson T. and Tunlid A. (1986) Recycling of fecal pellets in isopods: microorganisms and nitrogen compounds as potential food for Oniscus asellus L.. Soil Biology & Biochemistry 18, 595±600. Heilmann B. and Beese F. (1992) Miniaturized method to measure carbon dioxide production and biomass of soil microorganisms. Soil Science Society of America Journal 56, 596±598. Hopkins D. W. and Ferguson K. E. (1994) Substrate induced respiration in soil amended with dierent amino acid isomers. Applied Soil Ecology 1, 75±81. Hopkins D. W. and O'Dowd R. W. (1997) Chirality is a factor in substrate utilization. In Microbial Communities: Structural vs Functional Approaches, ed H. Insam and A. Rangger, pp. 215±228. Springer-Verlag, Berlin. Hopkins D. W., Isabella B. L. and Scott S. E. (1994) Relationship between microbial biomass and substrate induced respiration in soil amended with D- and L-isomers of amino acids. Soil Biology & Biochemistry 26, 1623±1627. Hopkin D. W., O'Dowd R. W. and Shiel R. S. (1997) Comparison of D- and L-amino acid metabolism in soils with diering microbial biomass and activity. Soil Biology & Biochemistry 29, 23±29. Kuhn J. and Somerville R. (1971) Mutant strains of Escherichia coli that use D-amino acids. Proceedings of the National Academy of Sciences (Washington), Vol. 68, pp. 2484±2487. O'Dowd R. W., Parsons R. and Hopkins D. W. (1997) Soil respiration induced by the D- and L- isomers of a range of amino acids. Soil Biology & Biochemistry 29, 1665±1671. Ryan B. F., Joiner B. L. and Ryan T. A. (1984) Minitab Handbook. Duxbury Press, Boston.
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