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Microbial biomass and metabolic activity in four acid soils
Biol. Biurhem. Vol. 10. No. 6. pp. Yl7-83. Printed m Great Britain. All nghts rcsrned
Soil
MICROBIAL
198X
Copyright
BIOMASS AND METABOLIC FOUR ACID SOILS M. DIAZ-RAMA,
Institute
de lnvestigaciones
0038-0717 YY 53.00 + 0.00 c I988 Pergamon Presr plc
ACTIVITY
IN
T. CARBALLAS and M. J. ACEA
Agrobiol&icas de Galicia (CSIC), Compostela. Spain (Accepted 25 March
Apartado
122,
15080
Santiago
de
1988)
Summary-The fumigation method was used to estimate microbial biomass C in four Haplumbrepts developed over different kinds of rock. In order to investigate the relationship between metabolic activity and microbial biomass and population density, CO, release from the glucose-enriched and unenriched soils
was measured during 28 days of incubation. Biomass C levels lay between 36 and I I2 mg
100 g-’ of dry soil, and made up only a small proportion of total soil C (0.77-1.38%). Only a small fraction of this biomass was detected by counting viables. but the microbial population was nevertheless significantly correlated with the biovass determined by fumigation. Among the physico-chemical properties of the soils, microbial biomass and population size were both chiefly affected (favourably) by humidity, total C and N and Al gel content. Metabolic activity was slight, either because part of the micro-organisms are inactive or because of a limited supply of substrate (the organic matter present may be unsuitable as a substrate or protected from microbial attack). Percentage C mineralization was inversely related to organic matter. silt and Al gel contents, and likewise failed to exhibit positive correlation with respiration. the biomass determined by fumigation or the counted population. The metabolic activity of the biomass appeared to depend upon the quality and nature of soil organic matter rather than its quantity, which nevertheless controlled microbial population size. Neither microbial biomass estimates nor viable population counts faithfully reflected metabolic activity in the soils.
INTRODIXTION
iological techniques (Anderson and Domsch, 1978; Smith et al., 1985) and extraction procedures (Jenkinson and Oddes, 1979: Webster et al., 1984). Though they all have their pros and cons, the fumigation methods appear to bc most reliable (Sparling, 1985) and their use has accordingly become widespread. COZ release is often employed as a measure of metabolic activity (Brookes, 1985; Feher, 1933; Golebiowska and Pedziwilk, 1984; Habcr, 3958, 1962; Koepf, 1952; Macfadycn, 1970; Witkamp and Frank. 1969), the assumption being that it derives mainly from the organic matter mineralized by heterotrophic micro-organisms. In the work described here, microbial biomass was estimated both by counting viables and by the fumigation method, and its metabolic activity was measured by respirometry.
Microbial biomass and its mctabolites constitute the active fraction of soil organic matter (Jansson. 1958). whose fast turnover shows its importance as a potential source of nutrients (Schniirer et al., 1985). Owing to their diversity, soil micro-organisms arc also able to perform numerous transformations under widely varying environmental conditions (Alexander, 1977); since these transformations are of great interest because of their effects on plant nutrition and growth, an understanding of the complex system constituted by the soil requires knowledge of microbial biomass and activity. Much effort has indeed been devoted to studying the relationships among the biomass and activities of the various soil microbial communities (Durska and Kaszubiak, 1983; Eiland, 198 I, 1985; Kaszubiak, 1986; Kaszubiak et al., 1977; Schniirer ef al., 1985), but successful research in this area is often hindered by the wide differences among micro-organisms as regards their ability to metabolize soil minerals or organic matter (Gray and Williams, 1971) and by the fact that any perturbation of the biological equilibrium of the soil alters the nature and rate of the metabolic processes in action and the release of CO, (Golebiowska and Pedziwilk, 1984). Microbial biomass has traditionally been estimated via biovolume by direct counting methods (Babiuk and Paul, 1970; Bratbak, 1985). but several indirect techniques have also been developed. These include various fumigation methods (Chaussod and Nicolardot, 1982; Jenkinson and Polwson, 1976). phys-
MATERIAL AND METHODS
Soils and sampling Samples were taken in April from the top 5-15 cm of the umbric horizon of four humid temperate zone soils classified as Haplumbrepts (Soil Taxonomy, 1975) (Profiles I I, 17, 33 and 3 I) developed over different kinds of parent rock (granite, acid schist, basic schist and gabbro, respectively) under different kinds of vegetation (pinewood. oakwood, pinewood and heath, respectively). The chief characteristics of these soils are listed in Table I. Between 30 and 50 samples were placed. in the field, in a sterile flask to prevent drying or heating. and immediately before 817
818
M. DIAZ-RAVICA er al. analysis this gross sample was sieved (< 2 mm) and the <2 mm fraction mixed thoroughly. For viable micro-organism counts, all sampling and sample manipulation was performed aseptically. Microbial biomass estimation Indirect method (fumigation). The method used differed from that of Jenkinson and Powlson (1976) as regards the incubation temperature and the determination of the CO: released. Three of six replicate 50g samples were fumigated with chloroform for 24 h before reinoculation with I g of fresh soil each. All six samples were then brought to 60% of field capacity and incubated for IO days in 500ml Erlenmeyer flasks placed in a water bath held at 22’C. The CO2 released was determined by evacuating it with a stream of moist. C02-less air that was then bubbled through NaOH solution of known strength which was subsequently titrated against HCI (Carballas et al., 1979). B, the quantity of microbial C, is given by B = F/K. where F is the difference between the quantity of CO2 given off by fumigated and unfumigated samples and K is the fraction of microbial C mineralized to CO: during the IO-day incubation. The value of K used. 0.4, was similar to that given by Anderson and Domsch (1978). who also incubated at 22C. Direct method (~~iablescounting). The total microbial population was estimated by counting (Clark, 1965). To do this 20 g samples were successively diluted x IO-‘-x IO-‘” in stcrilc water. Five tubes containing a liquid yeast extract and mineral salt medium (Clark. 1965; Wollum, 1982) were inoculated with each suspension, and populations were counted by the most probable number method (Alexander, 1965; Pochon and Tardieux, 1962). To convert the number of micro-organisms counted to g of microbial C it was assumed that the micro-organisms had a mean volume of I pm3, the value given by Alexander (1977) for bacteria, which are the predominant micro-organisms in these soils (Acea and Carballas, 1986), that their specific weight was I.1 g cm-‘; and that their dry matter content was 40%. 50% of which was C (Bratbak and Dundas, 1984). Microbial activity measurements In order to determine the metabolic activity of the microbial biomass, CO: release from glucoseenriched and unenriched soil samples with humidities of 60% of field capacity was monitored throughout 28 days of incubation at 22’C in a water bath. The technique employed was that developed by Carballas et al. (1979) after Guckert et al. (1968). A total of 32 Erlenmeyer flasks were incubated, I2 (3 replicates for each soil) containing 50 g of unenriched soil, I9 (5 replicates for each soil except one) containing 50 g of soil uniformly enriched with monohydrated glucose to a final value equivalent to 5% of total soil C and I empty flask used to check that the moist air stream employed during COr determination did not itself contain CO?. The CO? released as a consequence of C mineralization was measured at various times after the commencement of incubation using the same technique as in the fumigation experiments (see above). in order to compare the degrees of mineralization undergone in the four
Microbial biomass and metabolic activity soils, CC& release was related to total soil C by means of the coefficient of endogenous ~n~ral~tion (CEJM) and the coefficient of enriched soil mineralization (CESM), defined by CEM = ER/C,,, and where CESM = (TR - ER) x lO’/(C,, + C,,,), are to be expressed in mg, ER is the C,ti and C,,, endogenous respiration (the C given off by the unenriched soil, in mg) and TR is the total respiration (the C given off by the enriched soil, in mg). RESULTS AND DISCUSSION
Microbial biomass estimates Fumigation method. Table 2 shows that fumigated soil (FS) always released more COz than unfumigated soil (UFS). Even though these were acid soils, it was thus possible to use the fumigation method to estimate biomass C, since CO, flush [COr(FS) - COr(UFS)J was always positive. The opposite behaviour has been observed in soils with low pH by Powlson and Jenkinson (1976) and Williams and Sparling (1984). Biomass C, in the present soils, lay between 36 and 112 mg IOOg-’ of dry soil. Though these values are of the same order of magnitude as others that have been published (Azam et al., 1986; Jenkinson and Powlson, 1976), they amount to a smaller percentage of organic soil C, about 1%; and in keeping with the small proportion of microbial humin usually found in the acid soils of the region (Carbalias er al., 1983) they likewise mean that biomass C made up only a small fraction of total soil C. It may be pointed out that although the soils with least C also had least biomass, the latter constituted a greater proportion of total soil C than in the soils that were richer in C; a similar finding has been reported by Cerri and Jenkinson (1981). The variation in biomass C levels between the lowest (soil 17) and the highest (soil 31) may be explained in terms of their positive correlation with humidity, total N and totaIC(Tablcs I and2),es~ciailytotaiN(~ble3). Similar correlations were observed by Ros et al. (1980). Our soils also exhibited significant positive correlation between biomass C and Al gel content. In keeping with these correlations, soits 17 and 31 had, respectively, the lowest and highest levels of all these variables (Table I). Viables counts. The total microbial population densities of the soils studied, 106-IO’ micro-organisms g-’ of dry soil (Table 2), lie within the range reported by Acea and Carballas (1986) for a set of natural soils from the same region. The fact that the population of the soil developed over gabbros was far greater than those of the other three soils is explicable in terms of its high levels of humidity, total N, pH and Al gels, all of which are positively correlated with total microbial population (Table 3). and its low levei of ammoniacal N, which has a negative effect on microbial density. The same correlations have been observed in a study of 33 soils of the same region (Carballas et al., 1986). Most biomass C levels calculated by multiplying the number of bacteria by mean cell weight (Eiland, 1980) were less than I% of total biomass C as determined by the fumigation method (Table 2). a percentage just slightly less than others that have been published (Brookes, 1985: Eiland, 1980). The
819
Tabk 3. Corretation coeikient
(I) values among the biomass C, viable microorganisms and some characteristics of the soils
Soil property PH Moisture (%) Total C (%) Total N (%) A&O, CEM CESM Viable
Biomass C 0.7873 0.9756*** 0.8842. 0.991 I*** 0.965P’ -0.9768*** -0.9063* 0.887?*
Viable micro-organisms 0.9117. 0.8900. 0.6037 0.931v 0.9550’ 0.887?* -0.5555
CEM = Coefficient of endogenous mineralization. CESM = Coefficient of the enriched soil mineralization. l, l*, ***Significant at 0.05. 0.01 and 0.005 probability levels.
difference with respect to the fumigation figures is too great to be attributed to that part of the biomass made up by fungi that go uncounted because their growth in the culture medium is inhibited by its pH, or by other soil organisms that fail to grow even in the enriched medium (Clark, 1967). The counting and fumigation methods in fact measure different things: the fumigation method detects all soil microorganisms (bacteria, actinomycetes, fungi. algae and protozoa), whether or not they are actxve, whereas the counting method determines only that fraction of the total biomass which consists of viable microorganisms (Campbell, 1977; Kaczmarek et al., 1976). Indeed, according to Kaczmarek et al. (19731, counting mainly reflects the behaviour of zymogenous organisms that respond readily to the supply of organic substrate, and which make up only a small part of the total biomass. In the present study, the two measures were nevertheless significantly correlated at the 95% levet (r = 0.8872) the soils with greatest total biomass also having the greatest number of viable micro-organisms; and indeed, the relevant coefficents of correlation show that both these measures are chiefly determined by the same factors, total N, humidity and AI gel content. It is interesting that fumigation of soils I 1, 17 and 33, which had roughly equal microbial densities, increased release of CO, by a factor of about 2.5 with respect to the unfumigated soil, whereas CO? release by soil 3 I, the most densely populated, was increased some 3.5 times, presumably because of its having more dead micro-organisms. Microbiul activity
By the end of the 28-day incubation, unenriched soils had given off quite small quantities of COz (Fig. I), in spite of the ideal conditions for microbial activity. The greatest CO* release was by soil 1I, followed by soils 31 and 33, with soil 17 some way behind, though the differences were not statistically significant. CO, release was greatest during the first few days of incubation (Figs 2 and 3); except in soil 33, in which the process was slower, 7040% of the total quantity of CO1 had been given off by day 9 and 90% by day 16, by which time the mineralization process had levetled off. The addition of glucose stimulated respiration in all cases (Fig. l), the extent of the stimulation generally agreeing with the quantity of glucose added. This quantitative increase in mineralization appears to
M.
820
mgC/lOOg
DIAZ-RAVIRA
dry soil
er al
mgC/lOOg
dry soil
26
-
11
Fig. I. Cumulative CO: release from glucose-enriched (G) and unenriched soils during 28 days of incubation.
have been the only effect of the substrate, since the CO: release curves of the enriched soils exhibit the same ups and downs as those of the corresponding unenriched soils throughout the incubation (Figs 2 and 3). and the soil’s ordering by respiration value was unchanged by enrichment, though absolute differences were increased (Fig. 1). Because of the behaviour of soil 31, which had the greatest microbial biomass but low basal respiration, the whole group of four soils exhibited no significant correlation between endogenous respiration and biomass C as determined by the fumigation method, but the correlation was moderately significant (P < 0.1) when soil 3 I was excluded (r = 0.9471). The enriched soils behaved similarly (r = 0.9014, P < 0.1). However, neither the enriched nor the unenriched soils exhibited correlation between respiration and biomass C as determined by counting, showing that in spite of the claims made on its behalf (Babiuk and Paul, 1970), the counting method does not fdithfuhy reflect microbial metabolic activity. Whether enriched or not, it was the soils with the greatest organic matter contents that released most COz, the exception once more being soil 31, whose disproportionately small CO? production suggests the presence of some intrinsic mineralization-inhibiting factor in this soil. It may also be pointed out that comparison of CO? release by glucose-enriched and fumigated soils (Fig. 1 and Table 2) shows that dead microbial biomass had the same stimulatory effect on respiration as glucose, and is probably the most labile organic matter fraction (Jenkinson and Ladd. 1981). To sum up, the above findings show that in these soils the metabolic activity of the biomass is slight, possibly because part of the microbial population is inactive but also because of low effective substrate concentrations (especially in soil 31). The lack of effective substrate may be due either to the scarcity of immediately mineralizable material in these soils, or
-
17
-----
17+G
Fig. 2. Daily CO2 release from glucose enriched (G) and unenriched samples of soils I I and I7 during 28 days of incubation.
to such materials being in some way protected against microbial attack. The CEMs and CESMs calculated from respiration and total soil C values enable comparison of the degrees of mineralization in the four soils. Comparison of the CEMs shows (Fig. 4) that though the soils with the greatest organic matter contents released most CO,, they mineralized the smallest proportion of their C. In spite of its favourable conditions (pH, C-to-N ratio, biomass and viables count), soil 31 had the smallest CEM, followed by soil 11 (whose mineralization kinetics is almost parallel to that of soil 3 I but whose CEM is higher in spite of its unfavourable C-to-N ratio), soil 33 and soil 17. Furthermore, the CESMs show that the soils with the most intense endogenous mineralization were those in which mineralization was most stimulated by addition of glucose, soil I7 having the largest CESM, followed by soil 33 and a long way behind by soils 11 and 31. The evolution of the CESMs exhibited no coherent pattern during the first phase of the incu-
Microbial biomass and metabolic activity
bation period, but towards the end the CESM-time curves became parallel as the effect of the added substrate wore off as a result of its consumption or its retention in the soil in some unconsumable form. The inverse relationship between soil organic matter and degree of mineralization implies that the accumulation of the former, if it occurs, is due to an imbalance between the rate at which organic debris is supplied and the rate at which it is decomposed by mineralization. In turn, mineralization decreases as silt content rises, either because fine texture results in excessive water retention or because the micropores of fine-textured soils protect their organic matter from microbial attack (Adu and Oades, 1978a, b; Anderson and Paul, 1984). Another possible reason why mineralization was less intense in soils 11,3 1 and 33 than in soil 17 is their AI oxides contents, since although Al oxides do not prevent microbial growth, they may sequester organic matter from microbial attack by means of a physicochemical stabilization process (Carballas et al, 1979); this effect might be the cause of the striking results obtained for soil 31, an Haplumbrept over gabbros which in spite of its relatively large microbial population had very small mineralization coefficients.
811
1 1%
17
10 -
33
5-
r:i-:: .
0
_.--
__--
.’
0’
.*
,-
__
----
-4
__-
___----
11 --
mm_..________---
__-_--_
_/)
I
31 _-
-----
oays
3
5
,
9
14
fll
21
CESM
28
17+c
35-
30 -
25-
33+G
20 _________----15
__--
--
10
5
0 1
3
5
7
9
14
16
21
28
Fig. 4. Cumulative curves of the coefficient of endogenous mineraljzation (CEM) and the coefkient of enriched soil mineralization (CESM) during 28 days of incubation.
I
20
3
s
7
9
21
2
r
Fig. 3. Daily CO: release from glucose-enriched (G) and unenriched samples of soils 31 and 33 during 28 days of incubation.
There was no positive correlation between the coefficients of mineralization on the one hand and either respiration, viables counts or biomass as determined by fumigation on the other. Thus the least intense mineralization was exhibited by soil 31, which had large biomass and population values and respiration as good or better than that of soils with less biomass (soils 33 and 17); while soil 17, which had much less biomass than the others and slightly less respiration than soils 31 and 33, had the largest mineralization coefficients, either because a greater proportion of its biomass was active, or because the specific metabolic activity of the population was particularly intense, or possibly because the availability of substrates was less in the other soils. Since microbial biomas was positively influenced by two factors that showed a negative influence on the mineralization coefficients (totai C and Al oxides), it seems reasonable to conclude that the metabohc activity of the microbial biomass in these soils depends to a large extent on the quality and nature of soil organic matter rather than on the quantity microbial present, though the latter controls population density.
M. DIAZ-&%VI%A
822
Conclusions regarding microbial
biomass
and
acticyity
measurement
Though the fumigation method showed that all four soils had considerable quantities of biomass. the low COz release (ER) reflected low activity levels that were probably the result of a large proportion of the biomass remaining inactive (Lynch and Panting, 1981; Spariing, 1985). This ina~ti~ty may have been due both to lack of suitable organic substrate (Gray, 1976; Lynch, 1982; Paul and MacLaren, 1975; Paul and Voroney, 1980), since in all the soils the addition of readily degradable glucose produced an immediate rise in respiration (Anderson and Domsch, 1978), and to the substrate present having been protected from microbial attack, which would explain the differences in activity among the various soils studied. Thus neither high biomass nor high microbial population ensure that vigorous metabolic activity take place in the soil, and both population and activity must therefore be measured in order to determine and explain the behaviour of soil micro-organisms. The fact that endogenous respiration was more closely correlated with the biomass estimated by the fumigation method than with that estimated by counting implies that it is more useful to measure total biomass (by fumigation) than the fraction detected by counting. Acknowle&ernenrs-We thank Sr J. Caballo for drawing the figures and Sr Nieves and Sra Fernindez for typing the manuscript. This research was supported by Conselleria de Educati6n from the Xunta de Galicia.
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Williams B. L. and Sparling G. P. (1984) Extractable N and P in relation to microbial biomass in UK acid organic soils. Plum and Soil 76, 139-148. Witkamp M. and Frank M. L. (1969) Evolution of CO: from litter. humus and subsoil of a pine stand. Pedobiologia 9, 358-366.
Wollum A. G. II (1982) Cultural methods for soil microorganisms. In Methods of Soil Analysis Part 2. Chemical and Microbiological Properlies (A. L. Page, Ed.). pp. 781-802. American Society of Agronomy, Madison.
Soil
MICROBIAL
198X
Copyright
BIOMASS AND METABOLIC FOUR ACID SOILS M. DIAZ-RAMA,
Institute
de lnvestigaciones
0038-0717 YY 53.00 + 0.00 c I988 Pergamon Presr plc
ACTIVITY
IN
T. CARBALLAS and M. J. ACEA
Agrobiol&icas de Galicia (CSIC), Compostela. Spain (Accepted 25 March
Apartado
122,
15080
Santiago
de
1988)
Summary-The fumigation method was used to estimate microbial biomass C in four Haplumbrepts developed over different kinds of rock. In order to investigate the relationship between metabolic activity and microbial biomass and population density, CO, release from the glucose-enriched and unenriched soils
was measured during 28 days of incubation. Biomass C levels lay between 36 and I I2 mg
100 g-’ of dry soil, and made up only a small proportion of total soil C (0.77-1.38%). Only a small fraction of this biomass was detected by counting viables. but the microbial population was nevertheless significantly correlated with the biovass determined by fumigation. Among the physico-chemical properties of the soils, microbial biomass and population size were both chiefly affected (favourably) by humidity, total C and N and Al gel content. Metabolic activity was slight, either because part of the micro-organisms are inactive or because of a limited supply of substrate (the organic matter present may be unsuitable as a substrate or protected from microbial attack). Percentage C mineralization was inversely related to organic matter. silt and Al gel contents, and likewise failed to exhibit positive correlation with respiration. the biomass determined by fumigation or the counted population. The metabolic activity of the biomass appeared to depend upon the quality and nature of soil organic matter rather than its quantity, which nevertheless controlled microbial population size. Neither microbial biomass estimates nor viable population counts faithfully reflected metabolic activity in the soils.
INTRODIXTION
iological techniques (Anderson and Domsch, 1978; Smith et al., 1985) and extraction procedures (Jenkinson and Oddes, 1979: Webster et al., 1984). Though they all have their pros and cons, the fumigation methods appear to bc most reliable (Sparling, 1985) and their use has accordingly become widespread. COZ release is often employed as a measure of metabolic activity (Brookes, 1985; Feher, 1933; Golebiowska and Pedziwilk, 1984; Habcr, 3958, 1962; Koepf, 1952; Macfadycn, 1970; Witkamp and Frank. 1969), the assumption being that it derives mainly from the organic matter mineralized by heterotrophic micro-organisms. In the work described here, microbial biomass was estimated both by counting viables and by the fumigation method, and its metabolic activity was measured by respirometry.
Microbial biomass and its mctabolites constitute the active fraction of soil organic matter (Jansson. 1958). whose fast turnover shows its importance as a potential source of nutrients (Schniirer et al., 1985). Owing to their diversity, soil micro-organisms arc also able to perform numerous transformations under widely varying environmental conditions (Alexander, 1977); since these transformations are of great interest because of their effects on plant nutrition and growth, an understanding of the complex system constituted by the soil requires knowledge of microbial biomass and activity. Much effort has indeed been devoted to studying the relationships among the biomass and activities of the various soil microbial communities (Durska and Kaszubiak, 1983; Eiland, 198 I, 1985; Kaszubiak, 1986; Kaszubiak et al., 1977; Schniirer ef al., 1985), but successful research in this area is often hindered by the wide differences among micro-organisms as regards their ability to metabolize soil minerals or organic matter (Gray and Williams, 1971) and by the fact that any perturbation of the biological equilibrium of the soil alters the nature and rate of the metabolic processes in action and the release of CO, (Golebiowska and Pedziwilk, 1984). Microbial biomass has traditionally been estimated via biovolume by direct counting methods (Babiuk and Paul, 1970; Bratbak, 1985). but several indirect techniques have also been developed. These include various fumigation methods (Chaussod and Nicolardot, 1982; Jenkinson and Polwson, 1976). phys-
MATERIAL AND METHODS
Soils and sampling Samples were taken in April from the top 5-15 cm of the umbric horizon of four humid temperate zone soils classified as Haplumbrepts (Soil Taxonomy, 1975) (Profiles I I, 17, 33 and 3 I) developed over different kinds of parent rock (granite, acid schist, basic schist and gabbro, respectively) under different kinds of vegetation (pinewood. oakwood, pinewood and heath, respectively). The chief characteristics of these soils are listed in Table I. Between 30 and 50 samples were placed. in the field, in a sterile flask to prevent drying or heating. and immediately before 817
818
M. DIAZ-RAVICA er al. analysis this gross sample was sieved (< 2 mm) and the <2 mm fraction mixed thoroughly. For viable micro-organism counts, all sampling and sample manipulation was performed aseptically. Microbial biomass estimation Indirect method (fumigation). The method used differed from that of Jenkinson and Powlson (1976) as regards the incubation temperature and the determination of the CO: released. Three of six replicate 50g samples were fumigated with chloroform for 24 h before reinoculation with I g of fresh soil each. All six samples were then brought to 60% of field capacity and incubated for IO days in 500ml Erlenmeyer flasks placed in a water bath held at 22’C. The CO2 released was determined by evacuating it with a stream of moist. C02-less air that was then bubbled through NaOH solution of known strength which was subsequently titrated against HCI (Carballas et al., 1979). B, the quantity of microbial C, is given by B = F/K. where F is the difference between the quantity of CO2 given off by fumigated and unfumigated samples and K is the fraction of microbial C mineralized to CO: during the IO-day incubation. The value of K used. 0.4, was similar to that given by Anderson and Domsch (1978). who also incubated at 22C. Direct method (~~iablescounting). The total microbial population was estimated by counting (Clark, 1965). To do this 20 g samples were successively diluted x IO-‘-x IO-‘” in stcrilc water. Five tubes containing a liquid yeast extract and mineral salt medium (Clark. 1965; Wollum, 1982) were inoculated with each suspension, and populations were counted by the most probable number method (Alexander, 1965; Pochon and Tardieux, 1962). To convert the number of micro-organisms counted to g of microbial C it was assumed that the micro-organisms had a mean volume of I pm3, the value given by Alexander (1977) for bacteria, which are the predominant micro-organisms in these soils (Acea and Carballas, 1986), that their specific weight was I.1 g cm-‘; and that their dry matter content was 40%. 50% of which was C (Bratbak and Dundas, 1984). Microbial activity measurements In order to determine the metabolic activity of the microbial biomass, CO: release from glucoseenriched and unenriched soil samples with humidities of 60% of field capacity was monitored throughout 28 days of incubation at 22’C in a water bath. The technique employed was that developed by Carballas et al. (1979) after Guckert et al. (1968). A total of 32 Erlenmeyer flasks were incubated, I2 (3 replicates for each soil) containing 50 g of unenriched soil, I9 (5 replicates for each soil except one) containing 50 g of soil uniformly enriched with monohydrated glucose to a final value equivalent to 5% of total soil C and I empty flask used to check that the moist air stream employed during COr determination did not itself contain CO?. The CO? released as a consequence of C mineralization was measured at various times after the commencement of incubation using the same technique as in the fumigation experiments (see above). in order to compare the degrees of mineralization undergone in the four
Microbial biomass and metabolic activity soils, CC& release was related to total soil C by means of the coefficient of endogenous ~n~ral~tion (CEJM) and the coefficient of enriched soil mineralization (CESM), defined by CEM = ER/C,,, and where CESM = (TR - ER) x lO’/(C,, + C,,,), are to be expressed in mg, ER is the C,ti and C,,, endogenous respiration (the C given off by the unenriched soil, in mg) and TR is the total respiration (the C given off by the enriched soil, in mg). RESULTS AND DISCUSSION
Microbial biomass estimates Fumigation method. Table 2 shows that fumigated soil (FS) always released more COz than unfumigated soil (UFS). Even though these were acid soils, it was thus possible to use the fumigation method to estimate biomass C, since CO, flush [COr(FS) - COr(UFS)J was always positive. The opposite behaviour has been observed in soils with low pH by Powlson and Jenkinson (1976) and Williams and Sparling (1984). Biomass C, in the present soils, lay between 36 and 112 mg IOOg-’ of dry soil. Though these values are of the same order of magnitude as others that have been published (Azam et al., 1986; Jenkinson and Powlson, 1976), they amount to a smaller percentage of organic soil C, about 1%; and in keeping with the small proportion of microbial humin usually found in the acid soils of the region (Carbalias er al., 1983) they likewise mean that biomass C made up only a small fraction of total soil C. It may be pointed out that although the soils with least C also had least biomass, the latter constituted a greater proportion of total soil C than in the soils that were richer in C; a similar finding has been reported by Cerri and Jenkinson (1981). The variation in biomass C levels between the lowest (soil 17) and the highest (soil 31) may be explained in terms of their positive correlation with humidity, total N and totaIC(Tablcs I and2),es~ciailytotaiN(~ble3). Similar correlations were observed by Ros et al. (1980). Our soils also exhibited significant positive correlation between biomass C and Al gel content. In keeping with these correlations, soits 17 and 31 had, respectively, the lowest and highest levels of all these variables (Table I). Viables counts. The total microbial population densities of the soils studied, 106-IO’ micro-organisms g-’ of dry soil (Table 2), lie within the range reported by Acea and Carballas (1986) for a set of natural soils from the same region. The fact that the population of the soil developed over gabbros was far greater than those of the other three soils is explicable in terms of its high levels of humidity, total N, pH and Al gels, all of which are positively correlated with total microbial population (Table 3). and its low levei of ammoniacal N, which has a negative effect on microbial density. The same correlations have been observed in a study of 33 soils of the same region (Carballas et al., 1986). Most biomass C levels calculated by multiplying the number of bacteria by mean cell weight (Eiland, 1980) were less than I% of total biomass C as determined by the fumigation method (Table 2). a percentage just slightly less than others that have been published (Brookes, 1985: Eiland, 1980). The
819
Tabk 3. Corretation coeikient
(I) values among the biomass C, viable microorganisms and some characteristics of the soils
Soil property PH Moisture (%) Total C (%) Total N (%) A&O, CEM CESM Viable
Biomass C 0.7873 0.9756*** 0.8842. 0.991 I*** 0.965P’ -0.9768*** -0.9063* 0.887?*
Viable micro-organisms 0.9117. 0.8900. 0.6037 0.931v 0.9550’ 0.887?* -0.5555
CEM = Coefficient of endogenous mineralization. CESM = Coefficient of the enriched soil mineralization. l, l*, ***Significant at 0.05. 0.01 and 0.005 probability levels.
difference with respect to the fumigation figures is too great to be attributed to that part of the biomass made up by fungi that go uncounted because their growth in the culture medium is inhibited by its pH, or by other soil organisms that fail to grow even in the enriched medium (Clark, 1967). The counting and fumigation methods in fact measure different things: the fumigation method detects all soil microorganisms (bacteria, actinomycetes, fungi. algae and protozoa), whether or not they are actxve, whereas the counting method determines only that fraction of the total biomass which consists of viable microorganisms (Campbell, 1977; Kaczmarek et al., 1976). Indeed, according to Kaczmarek et al. (19731, counting mainly reflects the behaviour of zymogenous organisms that respond readily to the supply of organic substrate, and which make up only a small part of the total biomass. In the present study, the two measures were nevertheless significantly correlated at the 95% levet (r = 0.8872) the soils with greatest total biomass also having the greatest number of viable micro-organisms; and indeed, the relevant coefficents of correlation show that both these measures are chiefly determined by the same factors, total N, humidity and AI gel content. It is interesting that fumigation of soils I 1, 17 and 33, which had roughly equal microbial densities, increased release of CO, by a factor of about 2.5 with respect to the unfumigated soil, whereas CO? release by soil 3 I, the most densely populated, was increased some 3.5 times, presumably because of its having more dead micro-organisms. Microbiul activity
By the end of the 28-day incubation, unenriched soils had given off quite small quantities of COz (Fig. I), in spite of the ideal conditions for microbial activity. The greatest CO* release was by soil 1I, followed by soils 31 and 33, with soil 17 some way behind, though the differences were not statistically significant. CO, release was greatest during the first few days of incubation (Figs 2 and 3); except in soil 33, in which the process was slower, 7040% of the total quantity of CO1 had been given off by day 9 and 90% by day 16, by which time the mineralization process had levetled off. The addition of glucose stimulated respiration in all cases (Fig. l), the extent of the stimulation generally agreeing with the quantity of glucose added. This quantitative increase in mineralization appears to
M.
820
mgC/lOOg
DIAZ-RAVIRA
dry soil
er al
mgC/lOOg
dry soil
26
-
11
Fig. I. Cumulative CO: release from glucose-enriched (G) and unenriched soils during 28 days of incubation.
have been the only effect of the substrate, since the CO: release curves of the enriched soils exhibit the same ups and downs as those of the corresponding unenriched soils throughout the incubation (Figs 2 and 3). and the soil’s ordering by respiration value was unchanged by enrichment, though absolute differences were increased (Fig. 1). Because of the behaviour of soil 31, which had the greatest microbial biomass but low basal respiration, the whole group of four soils exhibited no significant correlation between endogenous respiration and biomass C as determined by the fumigation method, but the correlation was moderately significant (P < 0.1) when soil 3 I was excluded (r = 0.9471). The enriched soils behaved similarly (r = 0.9014, P < 0.1). However, neither the enriched nor the unenriched soils exhibited correlation between respiration and biomass C as determined by counting, showing that in spite of the claims made on its behalf (Babiuk and Paul, 1970), the counting method does not fdithfuhy reflect microbial metabolic activity. Whether enriched or not, it was the soils with the greatest organic matter contents that released most COz, the exception once more being soil 31, whose disproportionately small CO? production suggests the presence of some intrinsic mineralization-inhibiting factor in this soil. It may also be pointed out that comparison of CO? release by glucose-enriched and fumigated soils (Fig. 1 and Table 2) shows that dead microbial biomass had the same stimulatory effect on respiration as glucose, and is probably the most labile organic matter fraction (Jenkinson and Ladd. 1981). To sum up, the above findings show that in these soils the metabolic activity of the biomass is slight, possibly because part of the microbial population is inactive but also because of low effective substrate concentrations (especially in soil 31). The lack of effective substrate may be due either to the scarcity of immediately mineralizable material in these soils, or
-
17
-----
17+G
Fig. 2. Daily CO2 release from glucose enriched (G) and unenriched samples of soils I I and I7 during 28 days of incubation.
to such materials being in some way protected against microbial attack. The CEMs and CESMs calculated from respiration and total soil C values enable comparison of the degrees of mineralization in the four soils. Comparison of the CEMs shows (Fig. 4) that though the soils with the greatest organic matter contents released most CO,, they mineralized the smallest proportion of their C. In spite of its favourable conditions (pH, C-to-N ratio, biomass and viables count), soil 31 had the smallest CEM, followed by soil 11 (whose mineralization kinetics is almost parallel to that of soil 3 I but whose CEM is higher in spite of its unfavourable C-to-N ratio), soil 33 and soil 17. Furthermore, the CESMs show that the soils with the most intense endogenous mineralization were those in which mineralization was most stimulated by addition of glucose, soil I7 having the largest CESM, followed by soil 33 and a long way behind by soils 11 and 31. The evolution of the CESMs exhibited no coherent pattern during the first phase of the incu-
Microbial biomass and metabolic activity
bation period, but towards the end the CESM-time curves became parallel as the effect of the added substrate wore off as a result of its consumption or its retention in the soil in some unconsumable form. The inverse relationship between soil organic matter and degree of mineralization implies that the accumulation of the former, if it occurs, is due to an imbalance between the rate at which organic debris is supplied and the rate at which it is decomposed by mineralization. In turn, mineralization decreases as silt content rises, either because fine texture results in excessive water retention or because the micropores of fine-textured soils protect their organic matter from microbial attack (Adu and Oades, 1978a, b; Anderson and Paul, 1984). Another possible reason why mineralization was less intense in soils 11,3 1 and 33 than in soil 17 is their AI oxides contents, since although Al oxides do not prevent microbial growth, they may sequester organic matter from microbial attack by means of a physicochemical stabilization process (Carballas et al, 1979); this effect might be the cause of the striking results obtained for soil 31, an Haplumbrept over gabbros which in spite of its relatively large microbial population had very small mineralization coefficients.
811
1 1%
17
10 -
33
5-
r:i-:: .
0
_.--
__--
.’
0’
.*
,-
__
----
-4
__-
___----
11 --
mm_..________---
__-_--_
_/)
I
31 _-
-----
oays
3
5
,
9
14
fll
21
CESM
28
17+c
35-
30 -
25-
33+G
20 _________----15
__--
--
10
5
0 1
3
5
7
9
14
16
21
28
Fig. 4. Cumulative curves of the coefficient of endogenous mineraljzation (CEM) and the coefkient of enriched soil mineralization (CESM) during 28 days of incubation.
I
20
3
s
7
9
21
2
r
Fig. 3. Daily CO: release from glucose-enriched (G) and unenriched samples of soils 31 and 33 during 28 days of incubation.
There was no positive correlation between the coefficients of mineralization on the one hand and either respiration, viables counts or biomass as determined by fumigation on the other. Thus the least intense mineralization was exhibited by soil 31, which had large biomass and population values and respiration as good or better than that of soils with less biomass (soils 33 and 17); while soil 17, which had much less biomass than the others and slightly less respiration than soils 31 and 33, had the largest mineralization coefficients, either because a greater proportion of its biomass was active, or because the specific metabolic activity of the population was particularly intense, or possibly because the availability of substrates was less in the other soils. Since microbial biomas was positively influenced by two factors that showed a negative influence on the mineralization coefficients (totai C and Al oxides), it seems reasonable to conclude that the metabohc activity of the microbial biomass in these soils depends to a large extent on the quality and nature of soil organic matter rather than on the quantity microbial present, though the latter controls population density.
M. DIAZ-&%VI%A
822
Conclusions regarding microbial
biomass
and
acticyity
measurement
Though the fumigation method showed that all four soils had considerable quantities of biomass. the low COz release (ER) reflected low activity levels that were probably the result of a large proportion of the biomass remaining inactive (Lynch and Panting, 1981; Spariing, 1985). This ina~ti~ty may have been due both to lack of suitable organic substrate (Gray, 1976; Lynch, 1982; Paul and MacLaren, 1975; Paul and Voroney, 1980), since in all the soils the addition of readily degradable glucose produced an immediate rise in respiration (Anderson and Domsch, 1978), and to the substrate present having been protected from microbial attack, which would explain the differences in activity among the various soils studied. Thus neither high biomass nor high microbial population ensure that vigorous metabolic activity take place in the soil, and both population and activity must therefore be measured in order to determine and explain the behaviour of soil micro-organisms. The fact that endogenous respiration was more closely correlated with the biomass estimated by the fumigation method than with that estimated by counting implies that it is more useful to measure total biomass (by fumigation) than the fraction detected by counting. Acknowle&ernenrs-We thank Sr J. Caballo for drawing the figures and Sr Nieves and Sra Fernindez for typing the manuscript. This research was supported by Conselleria de Educati6n from the Xunta de Galicia.
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