Microbial biomass in relation to C and N mineralization during laboratory incubations

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MICROBIAL BIOMASS IN RELATION TO C AND N MINERALIZATION DURING LABORATORY INCUBATIONS K~RSTIN ROBERTSON’,JOHAN So*,

MARLWNECURHOLM~, TORBEXA. and THOMASR~SSWALL~

BONDE’

Department of Water in Environment and Society, Link6ping University, S-581 83 LiialriSpiag and *Department of Microbiology, Swedish University of Agricultural Seicnecs, S-750 07 Uppsala, Sweden (Accepred

30

September

1987)

Summary-Net carbon and nitrogen mincrahaation rates were dctcrmincd for an arable soil during 12 weeks at 37°C using an aerobic in~~tio~l~c~ng technique. The amounts of mineralized C and N were eomparcd to changes in the eontcnts of C and N in microbial biomass (as dctcrmincd by the chloroform fumigation incubation method, CFIM) during the incubation and to amounts of organic C and N in the Icaehates. Microorganisms were also followed by dircet counting of bacteria, mcasurcmcnts of total hyphal lengths and fluoresecin diaectate (FDA)-active hyphac. and by most probable number determinations of protozoa (naked amoebae and flagcllata). Numbers of naked amocbac increased nearly IO-fold initially and then deercased between weeks 6 and 12. Bacterial numbers and FDA-active hyphac dcercasc d during the incubation, and the relative composition changed slightly in favour of baetcria. Total hyphal hngths remained almost constant. A total of 105 Pg N g-’ soil dry wt and 1179 pg C g-’ soil dry wt was mineralized during the incubation, while the microbial N pool dcercascd by 42pgg-’ soil dry wt and the microbial C pool dcercased by 225 fig g-l soil dry wt. Soluble organic matter in the leachatcs amounted to 16 and 31% of mineralized C and N, rcspectivcly. The possibility of measuring C mineralization with less frequent lcachings and determinations of N mineralization offers an easy method for assessing changes in labile soil organic matter over time or for comparisons between soils.. Through the use of appropriate C-to-N ratios, the N-content in the labile pool

can he calculated.

lNTRODUClTON

The use of long-term, aerobic incubations to determine the potential ability of soils to mineralize nitrogen from organic matter, the N-mineralization potential (No), was first introduced by Stanford and Smith (1972). Since N, is considered to measure an important soil N source for plants (Stanford et al., 1973b). this incubation technique has been widely used to determine the effect of various agricuftural practices on soil fertility (Carter and Ret&e, 1982 El-Xaris et al., 1983; Griffin and Lame, 1983). In models simulating the dynamics of soil organic carbon and nitrogen the organic matter is usually separated into three to four fractions based on tumover rates, e.g. biomass active, non-biomass active, sfow and passive (Parton et al., 1983; Paul and Juma, 1981). The fractionation of the non-biomass organic matter is, however, rather arbitrary because of the lack of biologically relevant methods for determining pool sizes. Of the non-biomass fractions, only the active fraction has been determined experimentally thus far. i.e. by using isotopic dilution and curvepeeling techniques (Paul and Juma, 1981). A more straightforward approach would be the use of No as an estimate of this fraction, as suggested by Bonde and Rosswall (1987). Part of the microbial biomass might, however, be included in the measurement of N,. Despite extensive use of the long-term, aerobic incubation technique, not much is known about how

the extreme incubation conditions affect the microbial biomass. A close correlation between accumulation of mineral N and decrease in size of the microbial biomass N during a 12-week in~bat~on has, however, been observed (Carter and Rennie, 1982), and Bonde et al. (1988) have observed a marked initial decrease in biomass C. A slight decrease in microbial biomass N was also found by Paul and Juma (1981), but in their study the soi was not leached. Bonde and Rosswali (1987) bricAy discussed the biological implications of the course of N mineralization and hypothesized that a large part of the microbial biomass would decompose in a matter of weeks and that its relative composition would change in favour of fungi. Thus, microbial biomass seems to decrease during incubation, and this must be taken into account if the non-biomass active fraction is to be distinguished from that of microbial biomass. To simphfy the incubation technique, we tried to determine whether C mineralization could be measured as a complement to N mineralization. Since the mineralization of organic N is closely related to that of organic C (McGill et al., 1975). it was considered possible that the amounts of N mineralized wil1 be about equal to the amounts of C mincraliid muhiplied by a fixed N-to-C ratio. Potentially mineralizable N could then be quantified simply by determining respiration over time with only few leachings to avoid accumulation of N. WC thus determined the amounts of both carbon and nitrogen mineralized during incubation with a

modified Stanford and Smith (1972) technique (Bonde and Rosswall, 1987), and followed the changes in microbial biomass over time. Since Smith et al. (1980) observed that significant amounts of organic N can be leached along with the mineral N, organic C and N leached during the incubation-leaching procedure were also determined. MATERIAL AND METHODS Soil

were measured weekly with a Shimadzu GC-8A gas chromatograph fitted with a hot-wire detector. After the CO, measurement the tins were venti&& with compressed air. which was sufficient to decrease the CO, concentration to that of ambient air. Microbial biomass C and N

Carbon and nitrogen contents of the ~crobial biomass were determined by the chloroform fumigation incubation technique (CFIM; Jenkinson and Powlson, 1976). as modified by Schnfirer &al. (1983, every 2 weeks during the incubation. Determinations were made on fresh soif on the first occasion (time 0) and thereafter on soil-sand mixtures collected from the incubation before leaching. Approximately 20 g soil wet wt were used for each determination, Since the sieving of the soil might have caused increased respiration, the soil was incubated at 24°C for I week before the fumigation on the initial sampling (time 0). Biomass C and N contents were calculated with the following formula:

The soil was sampled from a long-term field experiment established in 1956 at the Swedish University of Agricultural Sciences, Uppsala. It is a sandy clay loam with a clay content of 35% and an original organic C content of 1.50% (Nilsson, 1980). When the soil samples were collected in 1986, the soil organic C content was 1.69%. the soil organic N, content 0.19%, and the pH 6.9 (Jan Persson, personal communication). The four replicate plots sampled had been annually cropped (generally with cereals), fertilized with 80 kg N ha-” yr” as Ca(NO,)r, and Bio-C = CO&-/O.4 1, straw incorporated at a rate equal to 1800 kg C ha-’ yr-’ (Jan Persson, personal communication). (Paul and Voroney, 1980) The straw was added every second year at twice the rate given above. The last addition was made in the and autumn of 1985, and the sampling was carried out in Bio-N = (NH:-N/NH~-N~~)~O.68, the beginning of April 1986. Three cores (3.2 cm dia) (as modified from Shen et al., 1984), were taken to a depth of 20cm from each of the four replicate plots. Cores from the same plot were where 1 denotes fumigated samples and bf denotes either mixed and sieved (2 mm) or air-dried and samples before fumigation. sieved (2 mm). All experiments and analyses were carried out on the four replicates, and the results are Microorganisms expressed per unit soil weight, regardless of later Bacteria were counted directly and their biomass admixtures with sand. calculated after size classification (Clarholm and Rosswall, 1980). Direct measurements of total hyphal N mineralilotion lengths and fluorescein diacetate (FDA)-active The procedure was based on the leaching method hyphae and estimates of most probable numbers of suggested by Stanford and Smith (1972) as modified protozoa were made (Schniirer el al., 1985) after 0,2, by Bonde and Rosswall (1987). Samples (50 g) of 6 and 12 weeks. air-dried soil were mixed with equal amounts of sand Chemical anatyses (grain size c2 mm), rewetted with 10 ml of distilled water, and gently mixed. The mixtures were transAmounts of inorganic N in soil extracts and ferred to polystyrene filter units (150 ml; Filter Unit leachates were determined by a flow injection analysis 7103, Falcon, Oxnard, Calif., U.S.A.) fitted with technique (Ruzicka and Hansen, 1981). using an cellulose-based filter membranes with 0.22 pm pore automated FIA star 5020 (Tecator AB, Hiiganls, size, and the soil was covered with two layers of fine Sweden). Nitrate and nitrite were analysed together mesh nylon nets (1 mm mesh size). After the addition and will be referred to as nitrate in the text. Total N of another 15 m1 of distilled water, soil moisture was in leachates was determined as described above after adjusted by means of a vacuum pump at an applied alkaline oxidation to nitrate with K,SrOE (Nydahl, suction of 33 kPa for 2.5 h (MacCay and Carefoot. 1978). Total organic C in leachates was determined 1981). on a flame ionization detector as CH, after catalytic The soil was leached with 200 ml IOrmt CaCI, oxidation to COr and then reduction to CH, (Larsson followed by 50ml minus-N nutrient solution (Stanand Lifjelund, 1980). ford and Smith, 1972) every 2 weeks for 12 weeks. During leaching the soil-sand mixtures were covered by glass-wool pads. The leaching rate was 250 ml h-’ RESULTS and the applied suction 13 kPa. After each leaching occasion soil moisture was adjusted as described Both C and N mineralization decreased sharply above, Between teachings the samples were held at between weeks 2 and 4 after initially high rates during 37°C. the first 2 weeks [Fig. I(a)]. After week 4 fairly constant minerali~tion rates were measured. The course of cumulative mineralization of C and The filter units containing the soil samples were N was the same for both elements, i.e. an initial flush held in gastight tins (2 1.) equipped with a rubber followed by a slower linear increase throughout the septum for gas sampling. Amounts of CO, produced

Microbial biomass and C and N mineralization

9.6

10.2

2

4

q5.0

16.4

t4.S

112

6

6

IO

12

wt. was 5.5% of soil organic N. This resulted in an overall C-to-N ratio of 11.2 for accumulated amounts. The ratio of mineralized C to N for each 2-week period increased until week 8 Fig. l(a)], after which it decreased, mainly because of increasing amounts of NH:-N in the leachates. The proportion of NO;-N to total inorganic N in leachates at the tirst sampling (99%) gradually decreased to 64% by the end of the incubation. On the two last leaching occasions (weeks 10 and 12) the filters were partly clogged, which caused flooding of the soils during the leaching procedure. This might have resulted in a more efficient extraction of mineral N, especially NH:-N, from the soil compared with the preceding dates, which in turn could have caused an apparent increase in the production of inorganic N. Consequently the C-to-N ratios on weeks 10 and 12 were possibly somewhat underestimated. In addition to inorganic N, the leachates were also analysed for organic C and N. Organic N in the leachate was 44% of the inorganic N on the 2nd week sampling. It then gradually decreased to about 20% by the end of the incubation (Table 1). Organic C was about 16% of the C0r-C produced on all dates.

Time (wk)

Total microbial

(b)

I

I

I

I

I

I

2

4

6

6

10

12

biomass

Total microbial biomass C, as determined by the CHCI,-fumigation technique (Table 2), decreased by 225 pg g-’ soil dry wt during the incubation, and by week 12 it was 49% of the initial amount. Biomass N decreased by 42 lug g-’ soil dry wt and levelled off at 35% of the initial N-content by week 8. The decrease in biomass C and N during the incubation accounted for 19 and 40%. respectively, of the measured amounts of C and N mineralized. Microbial respiration decreased to 32% of the initial rate by week 12. Bacteria, fungi and protozoa

Time (wk)

Fig. 1. (a) Carbon and nitrogen mineralization rates during a 12.week incubation of soil (mean values f SE. n = 4). (0) CO&, (a) NOT-N, (A) NH:-N. Values over x-axis denote C-to-N ratios of the produced inorganic C and N. (b) Accumulated amounts of mineralized C (@) and N (m) during a 12.week incubation of soil (mean values k SE, n =4).

[Fig. l(b)]. The straight-line phase of the mineralization course for C was, however, steeper than that for N. The amount of C mineralized, 1179 pg g-t soil dry wt. was 7.0% of soil organic C, and the amount of N mineralized, 105 pg g-’ soil dry incubation

Table 1. OrganicC and N

283

Total hyphal lengths remained almost constant throughout the incubation uabie 2). FDA-active hyphae markedly declined between weeks 2 and 6, and by week 12 they amounted to 26% of the initial length. At the start of the incubation, FDA-active hyphae accounted for 2.6% of total hyphal lengths and at the end they comprised 0.5%. Bacterial numbers declined during the incubation but not as rapidly as FDA-active fungi, and by week 12 they were 61% of the initial value (Table 2). Bacterial biomass decreased more rapidly than bacterial numbers, with the final value equal to 30% of the initial value. This was a consequence of a smaller average size of the bacteria during the later stages of the incubation.

in leachates(meanvaluesf SE. n = 4). Organic

N was calculated as total N minus

minaal N

Total N

OrganicC Incubation time (weeks) 2 4 6

a IO I2

OrganicN % of

% of IQ B-’ soil dry v/t 53 f I 26 f 2 27 f I 2s * I 26 + I 26:O

co,-c

wi* I 14* I Is*1 Is* I l6+ I l7II

Irg g-’ soil dry wt 51.3 * 24.9 f IS.0 * IS.3 f IS.3 t 14.8 E

2.1 I .7 1.7 0.7 0.6 0.7

CB g-’ soil dry wt 15.6 f 6.7 5 3.0 + 3.4 f 2.6 f 0.8 t

1.4 0.2 1.3 0.3 0.2 0.8

mineral N 44*5 38 f 2s* 28 f 20 f 7f6

3 I2 3 I

i@SRN

284

&mnso~

et al.

Table 2. Microbial charactcrirtica during the incubation (mean values + SE, n = 4) Incubation time (weeks)

Biomass

0

2

4

6

439 * 13

47s + 25

439 * 23

3t6+ 16

8

10

12

c

(jig C g-’ soil dry wt) Respiration’ (rg C g-’ soil dry WI) Biomass N (pg N g-’ soil dry wt) Total hyphal kngths (m g-I soil dry wt) x lo-’ FDA-act&c hyphae (m g-’ soil dry wt) Bacterial numbers (No. g-’ soil dry wt) x IO-* Bacterial biomass (pg dw g-’ soil dry wt) x IO-1 Amoebae (No. g-’ soil dry wt) x IO-’ RagelIatcs (No. g-’ soil dry wt) x IO-’ ‘Production of CO&

27Ok8

296* 17

214+ I2

54*3

49+4

47 f 4

37 * 2

116+5

81 fS

895 10

64i2

44+3

33 f 2

27 f: 2

23 k 2

26 + 2

22 2 I 1.22 + 0.29

0.90 * 0.09

1.16+0.09

ND

1.09+0.09

ND

ND

23 + 2

25 f 2

ND

513

ND

ND

6&l

6.9 & 0.9

1.6+ 1.i

ND

6.7 + 0.3

ND

ND

4.2 + 0.2

0.74 + 0.09

0.66 * 0.10

ND

0.66+ 1.10

ND

ND

0.22 * 0.03

1.3 * 0.5

8.9 * 2.0

ND

9.1 + 2.4

ND

ND

1.6 + 0.3

O.S*O.i

I.1 i:Oo.i

ND

Od8kOo.2

ND

ND

o/%4+0.1

in unfumigatcd samples during IO days of incubation.

Amoeba1 numbers increased nearly IO-fold during the first 2 weeks and then remained at hi& levels until week 6 (Table 2). At the end of the incubation period, fow numbers were again recorded. Flagellate numbers, which were lower than amoeba1 numbers, remained almost constant throughout the incubation. DISCUSSION

The standing crop values of the bacterial population provide little information about its activity. Bacterial production can, however, be related to the increase in naked amoebae, since they use bacteria as a major food source (Clarhofm, 1984; Elliott er al., 1984). Based on data in Cfarholm (I 98 I), the amount of bacteria required to cause the observed increase in naked amoebae between weeks 0 and 2 was calculated to be 190 pg dry wt g-j soil dry wt. The decrease in bacterial biomass during this time, however, only accounted for about 40% of this. The marked increase in naked amoebae during the early stages of the incubation (Table 2) thus indicated a high bacterial production during this period. After 6 weeks bacterial numbers and biomass decreased, most likely as a result of a decline in available substrate combined with predation by amoebae. Large numbers of protozoa would no longer be sustained by the lower level of bacterial production, and their populations consequently decreased. The reduction in FDA-active hyphae compared with total lengths and the decrease in the average size of bacterial cells indicated that energy was limiting for the microorganisms. A decrease in the proportion of FDA-active hyphae as a result of starvation was observed by Schniirer and Paustian (1988), and a sta~ation-i~du~d decrease in size has been found in bacteria (Amy and Morita, 1983). The decrease in FDA-active fungi occurred earlier during the incubation than did the reduction in bacterial biomass. Filamentous fungi are normally better adapted than unicellular bacteria for growth at low water potentials (Griffin, 1981). This competitive advantage is probably offset by the very high moisture conditions

employed during the Stanford incubation. The fungaf decrease might also have beu3n a result of the high tem~rature used during the incubation. The temperature was above optimum for growth of mesotermic fungi, which dominate in temperate soils (Griffin, 1972). In spite of the extreme incubation conditions, all estimations of microbial biomass and numbers were within the range of those reported for agricultural soils from this area (~hn~rer et ul., 1985, 1986). The initial biomass C value observed in this study (439 pg g-’ soil dry wt) was low compared with that reported earlier for the same soil. Bonde er al. (1988) for example, recorded a biomass C content of 812pgg-’ soil dry wt in April 1984, and in November 1982, a value of 604 pg g-’ soit dry wt was found by Schniirer et al. (1985). In addition, Bonde er al. (1988) detected a 51% decrease in biomass C during the first 4 wee&s of a similar incubation, whereas in our study no decrease was recorded during that period. It thus seems likely that part of the microbial biomass was killed as a result of the previous treatment of the soil (Powtson, f980) and then mineralized during the incubation period prior to the initial fumigation. The initial flush of mineralization (of both C and N) [Fig. l(a)] was probably due to the air-drying and sieving of the soil in combination with the temperature and moisture conditions of the incubation, which were near the optimum for ~neraIization. Air-drying and sieving render part of the soil organic matter more available to microorganisms owing to the destabilization of aggregate structures (Agarwal et al.. 1971; Powfson, 1980). Part of the microbial biomass is also killed by the treatment of the soil, and dead microbial cells are rapidly decomposed by surviving organisms (Jenkinson, 1976). The shift in the relative composition of inorganic N in the feachates towards NW:-N was probably caused by the temperature employed during the incubation, Possibly, 37’C is slightly above optimum for nitrification (Keeney and Bremner, 1967; Mafhi and McGiIf, I982), which thus was partially inhibited.

285

Microbial biomass and C and N mincraliition

The decrease in microbial biomass N during 12 weeks of incubation was equal to 40% of the N mineralized. This can be compared with 5% (recalculated using a k, of 0.68) during a 1Zweek incubation of non-leached soil samples at 28°C (Paul and Juma, 1981). Carter and Rennie (1982) found a close correlation between mineral N accumulation and decrease in microbial biomass N observed in a 12-week incubation-leaching experiment at 25°C. Their study also showed a relatively smaller decrease in biomass N compared with that obtained by us. The higher incubation temperature (37°C) thus seems to have caused a faster reduction of the microbial biomass. Normally, temperature optimum for mineralization is at least 37°C (Myers, 1975; Stanford er al., 1973a). The labile organic matter fraction was thereby depleted faster, and consequently, the microbial biomass decreased more rapidly during our incubation. The total amount of organic C and N in the leachates over 12 weeks was 16 and 3 1% of mineralized C and N, respectively (Table 1). In a similar incubation over 11 weeks at 35°C organic N in the leachates was about half of the leached mineral N (Smith er al., 1980). They assumed that this organic matter was readily mineralizable and obtained significantly different No values when calculated from total N leached rather than mineral N. The composition of this organic matter, i.e. whether it is readily mineralizable or not, determines whether it should be. included in the C, and N, fractions. By simply neglecting it, however, the soil C and N mineralization potentials can be seriously underestimated. The C-to-N ratios of the soluble organic matter in the 2 and 4 week leachings were of the same magnitude as that of microbial cytoplasm (McGill et al.. 1981). During the same period a marked decrease in microbial biomass was observed. It seems likely that this soluble organic matter is of microbial origin and thus it is probably easily decomposable. There is a close coupling between C and N cycling in the turnover of soil organic matter (McGill et al., 1975; Paul and Juma, 1981). This interdependence of C and N transformations is further reflected in the similarity of the cumulative C and N mineralization courses observed in this experiment [Fig. l(b)]. As C mineralization is easier to determine than that of N, it would be convenient to make more frequent C and less frequent N determinations. It must then be assumed that N immobilization is negligible in comparison with N mineralization or that the relative rates of these opposing processes remain constant. In our study it is likely that only small amounts of undecomposed plant material were present in the samples owing to the initial sieving of the soil, and net immobilization would consequently have been small. When comparing net C and N mineralization in two different tillage systems (zero and conventional) at four locations, Carter and Rennie (1982) observed a C-to-N ratio of 10.9 after 12 weeks of incubation. This is very close to the ratio of 11.2 obtained in our study. This similarity implies that in this kind of incubation, a C-to-N ratio near 11 is common for the organic fractions being mineralized in agricultural soils during the period considered. The use of respiration rates and a fixed C-to-N ratio instead of leach-

ings of N should, however, be used cautiously until tested more widely for different soils. Furthermore, accumulation of inorganic N must still be avoided by periodic leachings since the pattern of N mineralization was observed to differ between open (with periodic leaching of the soil) and closed system incubations (Maynard er al., 1983). Incubations should also be extended until the rates of accumulation of mineralized C and N have levelled off. A considerable amount of C and N mineralized during the 12-week incubation-leaching experiment could be accounted for in a decrease in the microbial biomass. If the labile pool of soil organic matter is to be distinguished from that of microbial biomass, the amounts of C and N mineralized will have to be adjusted with regard to this. In addition, organic matter in the leachates has to be considered, unless it is established that it is recalcitrant to microbial decomposition. The possibility of measuring C,, offers an easy method for quantifying the labile pool of soil organic matter, and through the use of appropriate C-to-N ratios its N-content can be calculated. Alternatively, regression models based on the C mineralization course can be used for calculating the N mineralization constant. Acknowledgements-We thank Professor Jan Persson for permission to use a long-term field trial established by the Department of Soil Science, Swedish University of Agricultural Sciences. The study was carried out as a joint project between the Department of Biology and the Department of Water in Environment and Society, University of Linkoping and the project ‘Ecology of Arable Land. The Role of Organisms in Nitrogen Cycling” at the Swedish University of Agricultural Sciences. Kerstin Robertson was responsible for the running of the experiments, chemical analyses and CFIM biomass, while Johan Schnrirer determined fungal biomass and Marianne Clarholm estimated bacterial and protozoan biomass. All authors arc qually responsible for the manuscript. REFERENCES

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