Enhanced nutrient mineralization and leaching from decomposing sitka spruce litter by enchytraeid worms

Soil Bid. Biochcm. Vol. 21. No. 2, pp. I&188. Printed in Great Bntain

1989

0038-0717;89 53.00 + 0.00 Pcrgamon Press plc

ENHANCED NUTRIENT MINERALIZATION AND LEACHING FROM DECOMPOSING SITKA SPRUCE LITTER BY ENCHYTRAEID WORMS B. L. WILLIAMS* and B. S. GRrFnTHst The Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB9 ZQJ, Scotland (Accepted 20 Ocrober 1988) Summary-Sitka

spruce litter was subjected to two freeze-thaw cycles and then packed into 40 glass leaching columns. Twenty columns were each inoculated with 200 enchytraeid worms isolated from fresh untreated litter and the remaining 20 used as controls. The columns were continuously leached with distilled Hz0 for I6 weeks at 20°C. Leachates were collected weekly and litter harvested after 0. 4, 8 and I6 weeks. Phosphate, K’, Car+ and Mgr+ started to leach immediately and by the end of I6 weeks the accumulated amounts of each element released were significantly greater (P c 0.05) in columns with worms than those without. Ammonium and nitrate did not appear in leachates until after 8 weeks. At the end of the experiment. significantly more (P < 0.001) mineral-N had been leached from the inoculated litter, 18.1 mg compared with 12.7 mg N column-’ in the uninoculated control. Amounts of mineral-N extracted from the litters were similar in both treatments but total net production of mineral-N was approx. 10% greater in the worm inoculated material. The N and P contents of the added worms were relatively small and the enhanced leaching was attributed to a grazing effect of the worms that reduced nutrient immobilization in the microbial biomass.

MATERIALS AND METHODS

INTRODUCTION

Enchytraeid worms inhabit decomposing coniferous litter and can be grown on these litters under certain

conditions (Griffiths and Alexander, 1986). The presence of enchytraeid worms significantly decreased the amount of Cal+ leached from decomposing oak-leaf litter but had no detectable effect on K+, NH: and NO; (Anderson et al., 1983). In leaching experiments with mixed coniferous litters, worms introduced on spruce litter proliferated concomitant with an increase in N mineralization and nitrification (B. L. Williams and C. E. Alexander, unpublished), but these effects could not be ascribed solely to the presence of the worms. In soils, enchytraeids make vatiable contributions to the fauna1 biomass ranging from 2.2 to 4.6 g dry wt m-r in acid organic peats (Coulson and Whittaker, 1978) and a maximum of I.2 g dry wt m-* in mineral grassland soils (McKercher er al., 1979). However, their contribution to nutrient turnover is probably much greater than that of their biomass (Persson, 1983). Hanlon and Anderson (1980) reported marked decreases in fungal biomass and greater increases in the bacterial standing crop of decomposing oak litter inoculated with macroarthropods. This has been attributed to grazing from microsites influencing microbiological activity (Anderson et al., 1983) which in turn could significantly alter nutrient dynamics. Our objective was to assess the role of enchytraeid worms in nutrient release, in particular the amounts and different forms of nitrogen, mineralized and leached from decomposing litter of Sitka spruce.

*Author to whom reprint requests should be addressed. tPresent address: Scottish Crop Research Institute, Invergowrie,

saa?I

?--A

Dundee

DD2

5DA,

Scotland.

Litter leaching columns

Loose, relatively undecomposed litter was collected at several points from beneath 40-year-old Sitka spruce (Picea sitchensis (Bong.) Cart.] at Banchory forest, Fetteresso, Kincardineshire, Scotland (Nat. Grid Ref. NO. 818869). planted on cultivated brown forest soil on the Strichen series (Glentworth and Muir, 1963) developed on till derived from acid schists. The plot had been fertilized in 1980, 1981 and 1982 (4 years before sampling) with 200 kg N ha-’ yr-’ as NH,NO,, 100 kg P ha-’ yr-’ as unground phosphate rock and I50 kg K ha-’ yr-’ as KCI. The bulked litter was screened to remove twigs and cones and then submitted to two freezethaw cycles (- I8 to 2OC) to kill worms. Forty leaching columns (Griffiths and Alexander, 1986) were filled with 100 g fresh wt, equivalent to 30.65 g dry matter, and 20 were each inoculated with 200 enchytraeid worms (unidentified) extracted from freshly collected unfrozen litter (O’Connor, 1962). All columns were then leached with distilled H,O (2 ml h-l) for I6 weeks at 2O’C. Leachates were collected weekly and 5 columns from each treatment were harvested and sampled after 4, 8, I2 and 16 weeks. Worm numbers

Worm numbers were estimated weekly in the leachates which were then filtered through glass fibre (Whatman GF/F) filters and at monthly intervals in the harvested litters using a wet-funnel technique (O’Connor, 1962). Litter analysis

Duplicate sub-samples (IO g fresh wt) from each of 5 replicate columns were extracted sequentially first

184

3.

L. WU.UAMS and 8. S. GRIFFITHS

with 100 ml 10 I’IIM CaCI, followed by 50 ml 1 M KCI. Calcium chloride extracts were analysed for NH:-N, (NO; + NO;)-N, PO:.‘-P and organic-N and KC1 extracts for NH:-N. Microbial biomass-C was determined by the glucose induced respiration method of Anderson and Domsch (1978) modified to estimate evolved CO2 by gas chromatography (Sparling et al., 1981). Microbial biomass-N and -P including N and P in the worms were estimated after 18 h fumigation with ethanolfree CHCI,, followed by immediate extraction with Lomb CaCI, after removal of CHCI, vapour by repeated evacuation (Williams and Sparling, 1984). Organic and NH.,+-N in the 10m,~ CaCI, extract was estimated as NH: after digesting 5 m aliquots with 1 ml cont. H,SO, containing 0.1% (w/v) Se. The digestion was carried out in two stages, heating for 30min at 110°C followed by I h at 33O’C and NH: in the digest estimated calorimetrically (Crooke and Simpson, 1971). The flushes of organic and NH:-N, and PO,-P concentrations in the CaCI, extracts were converted to estimates of biomass N and P using recovery factors of 0.54 for KX (Brookes er al., 1985) and 0.40 for ir, (Brookes er al., 1982). Litter was dried at 70’C to estimate its moisture content and, for analysis of total N, P etc., subsamples of the dried, milled. material (I 50 mg) were digested with a I : 1 mixture of cont. H2S0, containing 0.1% (w/v) Se and 30% (v/v) H,O, (Wall et al., 1975). Ammonium-N and PO,-P in the digests were determined calorimetrically (Crooke and Simpson, 1971; Murphy and Riley, 1962) and Ca’+, Mg’+, K+ and Na+ using spectrochemical methods. Chemical analysis of leachates and extracts

Concentrations of NH:-N were determined colorimetrically using a weakiy alkaline mixture of sodium salicylate containing sodium nitroprusside (Crooke and Simpson, 1971) and sodium dichtoroisocyanurate. Nitrate was estimated as NO; after reduction with copperized cadmium (Henriksen and SelmerOlsen, 1971). Nitrite was not determined separately. Total (organic and NH:)-N in the IO mM CaCI, extracts and leachates was estimated as NH,-N in acid digests as described above. Phosphate in leachates and extracts was estimated calorimetrically (Murphy and Riley, 1962). The pH of leachates was determined potentiometrically and converted to Hi concentrations. Concentrations of base cations, K+, Na’, Ga’+ and Mg’+ in leachates were determined spectro~hemically.

RESULTS

Worm numbers and biomass-C

The freeze-thaw cycles had effectively ehminated enchytraeids from the litter (Fig. 1) and only a small number survived which may have developed from cocoons. In the cohtmns inoculated with worms, numbers remained steady for 8 weeks and increased to 1404 column-’ between 8 and 16 weeks. Estimates of microbial biomass-C (Fig. 1) were unaffected by the addition of enchytraeids and were similar in both treatments. Values increased gradually from 0.31 up to 0.35 g C column-’ after 12 weeks and then decreased to 0.27 and 0.24 g C in control and inoculated columns, respectively. Nitrogen

Soluble organic nitrogen (N,) was leached from the beginning until week i I in the worm treatment and week 12 in the control when concentrations in the leachates fell below the level of detection [Fig. 2(a)]. The accumulated amounts were consistently, though not significantly, higher in columns that contained added worms. Amounts of organic-N extracted from the litters with 10 mM CaCI, showed an overall decrease over the 16 week period. At the start of the experiment the litters contained some exchangeable NH:-N which decreased during the first 8 weeks of the experiment [Fig. 2(b)]. The release of NH.,+-N into the leachates was slight until 8 weeks and by IO weeks the accumulated values were significantly greater (P c 0.05) in columns inoculated with worms than those without. Nitrate-N was detected in the leachates from week 10 onwards [Fig. 2(c)] and by 16 weeks significantly greater (P < 0.001) amounts were leached from the columns inoculated with wotms than from those without. Total amounts of mineral-N (NH: + NO;) leached [Fig. 2(d)] were also signifi~antIy greater (P c 0.001) in the presence than in the absence of worms. Estimates of microbial-N (Table 1) including the N in the enchytraeid worms increased significantly with time and although microbial-N was lower in the worm inoculated than the control columns at 16 weeks the difference was not significant. Phosphorus

Phosphate extracted from the litters decreased without showing differences between the treatments

Statistical analysis

The leachates from each of 5 replicate columns per treatment were analysed in duplicate weekly and fitter from each of 5 replicate columns per treatment was extracted and analysed in duplicate after 4,8 and 16 weeks. The accumulated amounts of each ion in the leachates on each of the 16 sampling ocasions was subject to a combined analysis of variance. Estimates of microbial-N and -P etc. in the harvested litters, on each of the 4 sampling occasions were also subjected to a combined analysis of variance.

Weeks

Fig. I. Microbial biomass C. gcolumn-‘, in control (0) and inoculated litter (a) and numbers of enchytraeid worms in control (A) and inoculated litter (A).

Nutrient leaching from coniferous litter

185

(b)

6\\ N”,+

ti \\

2

F

(cl

20

(d)

NO;

Minwol-N

16

0

i

4

6

12

0

16

4

6

12

16

Weeks Fig. 2. (a) Organic-N (N,) in leachates and in IO mr.t CaCI, extracts of litter, (b) NH: in leachates and in IO mht CaCI, + I MKCI extracts. (c) NO; in leachates and in IO mt.t CaCI, extracts. (d) mineral-N. NH,+ + NO;. Accumulated amounts; mg column-‘. in leachates, (A-A) control, (ALA) inoculated with worms. Amounts per extraction, mg column-‘. (O- --0) control, (O- - -0) inoculated with worms. Bars indicate SE.

(Fig. 3). In the leachates, Pod-P was present from the start of the experiment and by 13 weeks the amounts released from the worm inoculated columns were significantly greater (P < 0.001) than those in the control. Microbial-P including the P content of the enchytraeid worms increased sharply during the first 8 weeks (Table I) and by I6 weeks had decreased slightly. On this occasion the worm inoculated litters contained significantly less (P c 0.01) biomass-P than the control.

Cation leaching

All base cations measured, Ca*+. Mgr+, K+ and Na+ were leached continuously from the start of the experiment and the rate of accumulation was significantly linear (P c 0.001) with time in each case. Over the I6 week period significantly greater amounts of K+, Ca*+ and Mgr+ were leached from the worm inoculated columns than from the controls (Table 2). These differences occurred earlier with K+ (8 weeks) than either Ca*+ (I2 weeks) or Mg2+ (I4 weeks).

Table I. Mean contents, mg columne’, of microbial-N and -P (including the enchytraeid worms) in harvested litters continuously leached for 16 weeks at 2O’C

Weeks Treatment Nitrogen Control worms Phosphorus Control worms

0 43.2 24.S” 3.2 3.4Ns

4

8

16

SE I + j 132 d.f.)

52.7 53.3-

52.3 53.0NS

82.6 69.7’=

3.53

26.4 22.0-

28.4

24.6 16.0.

0.82

2a.oNs

NS denotes not significantly different from control. *Denotes significantly dit%rcnt from control at P < 0.01

B. L. WILLIAMS and 8. S. Grurrrnts

0

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I

I 4

2

I 6

I 6

I 10

I 12

I 14

L 16

Weeks

Fig. 3. Accumulated amounts (mg column-‘) of PO,-P leached from litters. (A-A) control, (A-A) inoculated. and PO,-P extracted with IO m.c! CaCI, from harvested litters, (O---O) control, (+---a) inoculated. Bars indicate SE.

Leachates from the worm inoculated treatments had higher pH values than the controls and accumulated amounts of H” were significantly greater (P < 0.01) in the unamended litters from 10 weeks onwards. DISCUSSiON

The quantities of C, N and P in the 200 enchytraeids added to the columns were insufficient to account for the enhanced leaching. The worms were equivalent to approx. 7.2 mg dry matter (Coulson and Whittaker, 1978) containing about 4 mg C, 0.8 mg N (Persson, 1983) and 23 pg P (McKercher et al., 1979). Failure to register on the estimates of biomass-C by the method of Anderson and Domsch (1978) could be attributed to the non-utililization of glucose by the worms. On the basis of the same average contents, the 1400 worms column-’ at the end of the experiment were equivalent to approx. 28 mg C, 2.5 mgN and 16Opg P. Although the worms would not register on the biomass-C values it would be expected that they would be affected by CHC13 fumigation contributing about 8 and l%, respectively, to the microbial-N and -P in the final harvest. The density of worms in the litter at the end of the experiment was equivalent to 173 x IO’ m-?, Table 2.

assuming 1.15 kg oven-dry litter m-? at the field site (H. G. Miller, personal communication), which is not abnormally high and within the range 80-200 x ldm-’ obtained by Coulson and Whittaker (1978) for eutrophic blanket bog peat. The pattern of N-leaching from spruce litter was very similar to that observed in previous experiments (B. L. Williams and C. E. Alexander, unpublished) even though the samples used in this work had been subjected to two freeze-thaw cycles and had been taken from fertilized plots. The relatively high amounts of extractable NH:-N, N, and PO:--P were partly consistent with the effects of residual P fertilizer and with the effects of freezing and thawing on litters and forest floor material (Arp et al., 1980). In this and in previous leaching column experiments (B. L. Williams and C. E. Alexander, unpublished), mineral-N appeared in the leachates after a lag of 8-9 weeks. During this time, amounts of exchangeable NH,+-N decreased concomitant with an increase in biomass-N and -P corresponding to assimilation and immobilization into the microbial biomass. The enchytraeids curtailed this period of immobilization by 7-10 days probably by changing the microbial biomass through grazing (Anderson et al., 1983). Previously, enchytraeids added to decomposing oak-leaf litter had no significant effect on N release (Anderson er al., 1983) possibly because the experiment lasted only 6 weeks compared with 16 in this study. The microbial biomass in acid forest soils is predominantly fungal (Ingham er al., 1986; Sparling and Williams, 1986) but there were no differences between the treatments in biomass-C values to suggest that fungal mycelium was susceptible to grazing by soil animals. However, the effects of grazing fungal material may have been masked by efficient replacement with bacterial biomass in the gut. Ingham et af. (1986) in a study of the effects of bactericides and fungicides on microbial activity in LFH material from beneath lodgepole pine ascribed N-immobilization mainly to bacteria and mineralization to fungi. This is not consistent however with the enhanced mineralization and leaching in this experiment being attributed to replacement of fungal by bacterial biomass concomitant with a reduction in immobilization. Adams (1986) showed that nitrification in the spruce litter at this site was stimulated by additions of peptone suggesting that the process was effected by heterotrophic organisms utilizing organic-N substrates. However, nitrification was not evident before I1 weeks had elapsed and solubie organic-N was present in the leachates throughout this period although the exact chemical form was unknown. Nevertheless, nitrification commenced soon after the build up of NH,-N which occurred earlier in the presence of enchytraeids. Rates of mineralization

Mean accumulatedamounts

Treatment Control worms SE (&) 118 d.f.

fmcquiv column-‘) of H’, K’, Na’. Ca’*, Mg’*, NH;, NO; and PO:- leached from columns during 16 weeks cominuous leaching with distilled water at 2O’C H’

K’

2.44 2.04’ 0.313

0.35 0.46’** 0.012

Na’ 0.60 0.61Ns 0.014

Ca”

Mn”

NH:

NO;

PO:-

3.62 3.77.0’ 0.050

0.50 0.53”’ 0.004

0.13 0.16. 0.009

0.78 I.l2”’ 0.035

IS.5 1.75** 0.036

l.**.***Treatmcnt differences signiticantly different from the control at P < 0.05. c. 0.01 and < 0.001, respectively. NS denotes not significant from control.

Nutrient leaching from coniferous litter

were 12% higher and nitrification increased by 24%, in the worm inoculated litter compared with the control indicating that these processes were enhanced and not impaired by the presence of enchytraeids. This net increase in mineral-N, equivalent to about 2 kg N ha-‘, appeared to be at the expense of the N content of the microbial biomass although the differences between the treatments in microbial-N were not significant. Both biomass-N and -P were calculated using values for the recovery factors (KN and Kp) that had been derived using mineral soils fumigated with CHCI, and extracted with K2S0, (Brookes et al., 1985) and NaHCO, (Brookes et of., 1982). respectively. These recovery factors could vary with the nature of the biomass and could have changed during the course of the leaching experiment. Moreover, CHCI, fumigation may have extracted non-biomass N and P from the needle litter (McLaughlin and Alston, 1985) so that the estimates of microbial-N and -P should only be regarded as approximate. The incorporation of P into the microbial biomass was comparable with the amounts leached and together they accounted for a very high proportion of the total-P, i.e. 70%. There was probably sufficient P to meet the requirements of the changing biomass because the site had been fertilized. Enchytraeids increased overall amounts of PO:--P and increased net mineralization by about 10% with a simultaneous decrease in microbial-P. Biomass-C did not alter in the same manner as biomass-N or -P and C:N:P ratios starting at 96: 11: 1 changed to 10:3: 1 for the control and 17:4: I for the enchytraeid treatment after 16 weeks. A comparison of these values with those obtained by Anderson and Domsch (1980) i.e. 17:3: I for bacteria and 10:2: I for fungi, suggests that the enchytraeids have generated a predominantly bacterial biomass. This is consistent with the hypothesis that the fungal material is susceptible to grazing by the worms and that bacterial biomass in the animal’s

gut is enhanced

(Anderson

et al.,

1983). In contrast to the inhibition of Ca’+ leaching from decomposing oak leaves (Anderson et al., 1983) enchytraeids accelerated leaching of Ca*+, Md+ and K+ from the spruce litter. Differences in substrates and in the experimental conditions probably explain the different results. The relatively high amounts of Cal+ and K+ leached from the spruce litter reflect the fertilizer treatment 4 years previously. Total amounts of Cal+, M g’+ and KC leached during the 16 weeks were equivalent to 40, 43 and 21%, respectively, of the total contents of each element in the litter. Inoculating with enchytraeids increased these values to 42, 46 and 28%, respectively. The mean weight of litter on the forest floor at the site was approx. 1I.5 tonnes ha-’ (H. G. Miller, personal communication) and the amounts of each element leached corresponded to approx. 68 kg CaZc ha-‘, 6 kg Mg’+ ha-’ and 33 kg K+ ha-‘. The presence of the enchytraeids raised these values by 1.4 kg Ca’+ ha-‘, 0.2 kg Mgz+ ha-’ and 2.3 kg K+ ha-’ so that the worms had their greatest effect on K’. The enhanced release of all base cations reduced the acidity of leachates and this could have important consequences in situ though it is uncertain that the worms would behave similarly

187

if leached with throughfall and stemflow waters enriched with cations and anions. In conclusion, it has been shown that the enchytraeids had a marked effect on the partitioning of N and P between leachates, exchangeable forms and microbial biomass. These changes occurred mainly during the last 8 weeks of the experiment when worm numbers were still increasing. Worm numbers were still increasing when the experiment ended but previous experiments (B. L. Williams and C. E. Alexander, unpublished results) have indicated that mineral-N would continue to be released after worm numbers had reached a maximum. Although the enchytraeids had a relatively small effect on the amounts of N mineralized during the incubation the enhanced microbial activity and turnover of biomass could have a greater significance at the lower temperatures and moisture contents obtained in the field. Acknowledgemenrs-We

thank the Conservator (North Scotland), Forestry Commission, for permission to take

samples at the site and Miss Miriam Young and Miss Angela Smith for technical assistance.

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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 IO,21 5-22 1. Anderson J. P. E. and Domsch K. H. (1980) Quantities of plant nutrients in the microbial biomass of selected soils. Soil Science 130, 21 l-216. Anderson J. M., Ineson P. and Huish S. A. (1983) Nitrogen and cation mobilization by soil fauna feeding on leaf litter and soil organic matter from deciduous woodlands. Soil Biology & Biochemistry 15, 463-467.

At-p P. A., King H. B. and Krause H. H. (1980) Storage effects on nutrient mineralization in coniferous forest floor samples. Canadian Journal of Soil Science 60, 5 17-525.

Brookes P. C., Powlson D. S. and Jenkinson D. S. (1982) Measurement of microbial biomass phosphorus in soil. Soil Biology & Biochemistry 14, 319-329.

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from soil. In Progress in Soil Zoology (P, W. Murphy, Ed.), .no. . 279-285. Butterworths. London. Persson T. (1983) influence of soil animals on nitrogen mineralization in a northern Scats pine forest. In Proceedings warmth Internario~~ Co~Io~uium on Soil Zoology (T. P. Lebrun, Ed.}. pp. 117-126. Sparling G. P. and Williams B. L. (1986) Microbial biomass in organic soils: estimation of biomass C, and effect of glucose or cellulose amendments on the amounts of N and P released by fumigation. Soil Biology & Biochemisrry 18, 507-513.

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