Nitrogen transformations in soil as affected by bacterial-microfaunal interactions

CQ38-07I 7/82~02~3-~~3.~/5 Copyright B 1982 Pergirmon Press Ltd

Soil Bhd. Bitwkm. Vol. 14. pp. Y3 to Y8. 1982 Prinlcd in Great Britain. All rights reserved

NITROGEN TRANSFORMATIONS BY BACTERIAL-MICROFAUNAL

IN SOIL AS AFFECTED INTERACTIONS

L. E. WOODS*,.C.V. COLE,’ E. T. ELLIWM, R. V. ANDER~QN and D. C. COLEMAN* Natural Resource Ecology Laboratory,

*USDA-SEA Faculty Affiliate and ‘Department of Zoology/Entomology. Colorado State University, Fort Colhns, CO 80523, U.S.A. (Accepted 10 August 1981) Summary-We investigated the effects of soil microfauna feeding on a soil bacterium ~~se~d~~fl~s cepaciu) and the resultant influence on net N mineralization. As the bacteria1 biomass increased, it assimilated N from the soil. Later, only if this bacterial biomass decreased was N remineralized. Grazing by amoebae (Acanthamoeba pofyphaga) always reduced bacterial biomass, increased respiration, and increased nitrogen mineralization. Grazing by nematodes (~es~djpfogaste~ lheritieri) always reduced bacterial numbers and increased respiration, but only increased N mineralization when nematode populations themselves declined from peak values. A N budget calculated for the populations indicated that the nematode biomass was not sufficient to account for the unmineralized N, so we postulate change in excretory pathways as the bacterial food becomes limiting. This budget further indicated that amoeba1 and bacterial biomass could account for all of the non-mineral N when only these two species were present. Our experiments showed that microfauna can play an important role in N mineralization in soil and that the mechanism for this role is more likely to be through direct excretion by the grazers than through indirect physiological effects on the bacteria.

INTRODUCTION In

soil, plants and microorganisms depend on each other for nitrogen and carbon respectively. In these flows of C and N, organisms of several trophic levels interact. Both protozoa and nematodes are common in soils and can have large populations (Elliott and Coleman, 1977; Darbyshire, 1975; Yeates, 1979). Feeding on bacteria and fungi by these populations may perform a substantial role in a key process of the N cycle-mineralization. Members of our group have investigated microbiological details of soil chemical tr~sformations. We reported previously the biological interactions (Anderson et al., 1978), and the relationship of these interactions to C (Coleman et nl., 1978) and P transformations (Cole et af., 1978). We now report the relationship of these trophic interactions to N transformations. Johannes (1968) reported that mineralization of N and P from organic matter in aquatic systems is strongly dependent on grazing of the bacterial populations by microfauna. He differed with previous workers in emphasizing the microfaunal role in nutrient regeneration. He stated that this lack of emphasis was “ . . . based on the supposition that standard nutrient cycles worked out for soil apply a priori to water as well.” These nutrient cycles may have been over-simplified for soil in the first place. While bacteria are the first decomposers in aquatic systems (Golterman, 1976), bacterial-fading organisms are major agents in mineralization that is not ac*Present address: USDA/ARS, 8408 Hildreth

Rd,

Cheyenne, WY 82001, U.S.A. s&a.

14/z--a

93

complished by bacteria alone (Johannes, 1968). Both bacteria and fungi are primary decomposers in soils (Clark, 1969), but, here as well, bacterial-feeders may enhance mineralization, as was true for P in our experiments (Cole ef al., 1978). The mechanism of this enhancement can be either stimulation of the primary decomposers or direct excretion from the mierobivorous fauna. We sterilized soil and added (singly, or in appropriate combinations) inocula of pure cultures of bacteria, amoebae, and nematodes. While this in no way represented the population of a field soil, it did two things: First, those interactions we wished to study, and only those interactions, occurred in any one flask. Second, we were able to use bacteria readily counted by the dilution plate method as many soil bacteria are not. Coleman et al. (1977) reported increased release of mineral N and P as well as respired C in soil microcosms with gnotobiotic populations of bacteria with either amoebae or nematodes compared with’ bacteria alone. In the present series of experiments, we had up to three trophic levels (the nematode Mesodiplogaster feeding on bacteria and amoebae), two levels of added C, and incubations of up to 65 days. Amoebae have two life-stages, either cystic or trophic, whereas nematodes pass from eggs through a series of immature stages to adults. The nematode populations declined at different times in these experiments, allowing us to observe the relationship of their population status (in~easin~ maint~nin~ or declining) to N and C mineralization. These population processes are common in soils and we present evidence here that they affect N mineralization.

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Neither amoebae nor nematodes would grow in this medium without the bacteria present or more nutrients added. Amoebae were raised axenically on the same medium, with 13; proteose peptone, l”/:, glucose, and a phosphate buffer. Amoebae grown on this medium were aseptically washed before inoculation, concentrated by centrifugation, and inoculated into the soil microcosms when in log phase at a density of MATERIALS AND METHODS approximately 1 x 104g-’ soil. Stock cultures of the We wilI describe N transformations in three experinematodes were maintained on nutrient agar plates ments. The first experiment had four biological treatwith P. cepuciu as the sole food organism. Prepments, each with two glucose concentrations and aration of these nematodes for inoculation into microcosms involved washing them off the agar plates lasted for 24 days. There were five sample days, after 3, 4, 7, 17 and 24 days of incubation. The biological and surface sterilization with merthiolate (1: 1000 treatments were: bacteria (~seu~u~o~~s cepacia) dilution) for 3 min. Merthiolate was removed by five alone, bacteria plus either amoebae (~~cunr~a~e~~ washings with sterile water. Nematodes were then pol~p~ugo), or nematodes (~esodip~o~asfer ~~erir~eri), concentrated by ~entrifugation and added to microand all three species together. Carbon treatments cosms at 10 nematodes g-’ soil in nematode treatwere added on days 0, 3 and 6. They were: (1) ments. In all experiments we used flasks treated with amended (200 pg glucose-C per g- ’ dry soil in 1 ml of all amendments but without inocula to determine misolution) and (2) unamended (three equal volumes of crobial contamination and initial N and P concensolution without glucose). Amended treatments then trations. received a total of 600 pg C g- ’ dry soil. In all experiments, the soil remained near field caThe second experiment ended with a single sampacity moisture (150,). In our flasks and respiratory chambers, very little moisture was lost and we did not pling at 14 days. The biological treatments were the same as the first experiment, but 5OOpg glucose-C add any water, even in the long term experiment. When sampled the soil was thoroughly mixed and g - ’ soil was added at the beginning of the incubation. subsampled for the various analyses. Bacteria were The third experiment had three biological treatments: bacteria alone, and two different nematodes counted by the dilution plate technique with 3 repli(Acrobeloides sp. and ~esod~plo~osfer l~eririeri) feedcates of the t06 through 10” dilutions plated onto nutrient agar. Amoebae were counted using Cooke ing on bacteria. Five hundred pg glucose-C and 50 pg microtiter dilution plates (8 repticate wells) and the NH:-N g- ’ soil were added with the inoculum. most probable number technique (Darbyshire et ~1.. There were two sample dates after 10 and 65 days of 1974). Nematodes were extracted by a modification of incubation. For other aspects of this experiment see Anderson and Coleman (1981) and Anderson et al. the Baermann technique (Anderson er uI., 1979), followed by counting concentrated extracts on grid (1981). We used Olney sandy loam soil (mixed, mesic, counting plates. To calculate production of the biota, we converted ustoiiic, haplargid) collected from the Pawnee Site from ~pulations (numbers) to dry weight, and then (the field site of the Natural Resource Ecology Laborto carbon. Bacteria populations were estimated by atory, Colorado State University, located on the plate counts and size-class distribution measured USDA, Science and Education Administration Cenfrom soil suspensions using a Filar micrometer ocutral Plains Experimental Range near Nunn, Colorlar. Amoeba1 cell volumes were measured with a Filar ado). We air-dried and sieved the soil (maximum parmicrometer, and individual cell dry weights were calticle size 1 mm). Twenty grams of the air-dry soil in culated as 1.2 mg individual - I. Nematode biomass SOmI conical flasks were wetted to field capacity was determined using volume-specific gravity tech(roughly 15% moisture), incubated for 2 days to estabniques (Overgaard-Nielsen, 1949). The wet weights of lish microbial activity. Then the flasks of soil were adult females were 1.9 pg individual - 1 and for dauer plugged with cotton, sterilized by placing in a sealed larvae 0.9 pg. The dauer larvae are immature stages chamber, evacuating the chamber, and introducing propylene oxide until the chamber atmosphere was that retain the cuticle from the preceding larval stage: this is a useful survival mechanism in times of stress. saturated. After 48 h, the sterilized flasks were air Dry weight was calculated as 207: of wet weight, and dried in a forced-draft oven at 35°C for 2 weeks. ProC as SOP.;of dry weight. pylene oxide fumigation added roughly 1600 pg g- ’ NH: -N was extracted from the soil by shaking 5 g residual propylene glycol C to the soil (Anderson er al., 1978) which added to the available substrate for soil in 50 ml 2 N KC1 on a platform shaker for 30 min (Bremner, 1965). The extract was filtered and the the inoculated bacteria, NH:-N concentration determined using modified We isolated the bacteria, amoebae and nematodes Conway microdiffusion dishes (Stanford et al., 1973). from near where we coiiected the soil. Bacteria and Nitrate concentrations were very low ( < 1 gg N g- ‘) amoebae were isolated from the rhizosphere of blue and as no nitrifiers were present, none was produced; grama, Bouteloua grucilis (H.B.K.) Griffiths, based on for this reason NO; values are not included in this the method of Louw and Webley (1959). Nematodes report. were extracted with a Baermann funnel technique and The flasks were treated throughout as randomized cultures started with gravid females. Pure cultures of blocks for replication. Three replicates of each treatbacteria (P. cepacia) were grown in a reference soil ment were destructively sampled on each sample date. solution medium (RSS; Herzberg et af., 1978) with All differences reported are significant at least at the 0.2% glucose, 1 mg I- ’ Fe and 50 mg I- ’ asparagine. Our purposes were to: (a) vary biological complexity and note its effect on N mineralization, (b) evaluate popuIation responses to glucose amendments in terms of N mineralization, and (c) evaluate N flow as related to C distribution.

95

N transformations and microfauna

0

34

7

i7 Time,

0

24

34

7

17

24

Time, days

doyi

Fig. 1. The effect of the trophic interactions of bacteria, amoebae, and nematodes on the amounts of NH:-N in the first experiment without added glucose. O-----O Bacteria (B); 6---O Bacteria, Amoebae (BA): A---A Bacteria, Nematodes (BNI: V... V Bacteria. Amoebae. Nematodes (BAN)

95’:” confidence level and means separated by Tukey’s Honestly Significant Differences.

RESULTS During the early phases of bacterial growth, N was rapidly taken up by the bacteria, regardless of the biological ensemble or glucose concentration (Figs 1 and 2). In the first experiment, NHf-N concentrations dropped from 47 pg NH:-N g-’ soil to 7.4 or less in 3 days. Throughout the 24 days of this experiment, bacterial activity alone did not result in a net mineralization of N; amoebae feeding on the bacteria reduced bacterial numbers (Anderson er af., 1978) and, at the same time, miner~ized 32pg NH;-N g- ’ soil. Nematodes feeding on bacteria afso reduced bacterial numbers, but did not mineralize any N, though they increased respiration (Coleman et al., 1978). Nematodes, feeding on bacteria and amoebae together, reduced numbers of both, and mineralized an intermediate amount of N (16~4g N g-’ soil). Nematodes reduced the numbers and the effectiveness of amoebae in N mineralization. Both nematodes and amoebae increased respiration of C (Coleman et ul. 1978); but Cole et al. (1978) found that, similar to N, only amoebae were effective in mineralizing P. Nematodes were at peak populations or had only slightly declined at the end of this first experiment, and may

Fig. 2. The effect of trophic interactions of bacteria, amoebae and nematodes on the concentrations of NH*+-Nin the first experiment with added glucose-C (6OD@gC/g soil). Symbols same as those of Fig. 1.

have excreted organic forms of N. As we shall show later, if N was retained by the nematode population, its carbon to nitrogen (C: N) ratio would be unreasonably low (0.2). Only treatments without added glucose mineralized N (Fig. 1). Only amoebae with bacteria produced measurable NH: in the presence of added glucose, but not significantly (P = 0.05) more than the other treatments (Fig. 2). In the second experiment, after 14 days amoebae with bacteria mineralized 32.7 pg NH:-N g- * (Table 1). Again in this case, nematodes did not mineralize more N than bacteria alone. There were 50,ug NH:-N g-’ in this soil after it was sterilized. It is probable that patterns of uptake and remineralization similar to the first experiment occurred in the first few days of this experiment. Since the nematode (M. /heritieri) has a generation time of 4 days (Anderson and Coleman, 1981), the ~pulation status after 14 days should be similar to the first experiment; either at peak values or nearly so, in any case not declining. In the third experiment, incubated for 65 days, the nematode population declined. In this experiment we compared two nematodes (the same M. lheririeri and an Acrobeloidessp.) with bacteria alone. After 10 days bacteria took up nearly all of the NH:-N (down to 2 pg NH:-N g-’ soil). In this case, after only 10 days both nematodes mineralized more nitrogen than bacteria alone (Table 2). After 65 days bacteria alone mineralized 35 fig NH:-N g-‘; but both nematodes mineralized 50 pg NH:-N g- ’ soil.

Table 1. The effectof amoebae and nematodes feeding on bacteria on the concentration of NH:-N in soil after 14 days of incubation Biological treatment

pg NHf-N g-’ soil

Bacteria (B) Bacteria, Amoebae (BA) Bacteria, Nematodes (BN) Bacteria, Amoebae, Nematodes (BAN) * Significantly different at the a = 0.01 level.

20.9 32.7+ 18.3 19.9

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Table 2. The effect of two nematode species on the amount of NH:-N in soil in the third (65-day) experiment Biological treatment

Days

Bacteria

Nematode Biomass-C

NH,+-N

Predicted* NH,+-N

IO 65

0 0

pg g-’ 2 35

NDI NDP

10 65 10 65

35 20 22 7

18 51 22 49

62 65 46 49

Nematodes and bacteria (Acroheloides) (Mesodiployoster)

* Assuming 150 pg NH:-N present initially, 50 peg(NH&SO,-N _ ‘g were added to the same soil used for Table 4, and correcting bacterial biomass to account for 500 pg glucose-C added instead of 600 pg in the BAN experiment. t Bacterial numbers not determined.

DISCUSSION

soil after 24 days when not grazed. We further esti-

Amoebae feeding on bacteria consistently increased N mineralization in all experiments. In the first and second experiments amoebae with bacteria mineralized more N than either bacteria alone or nematodes with bacteria. Elliott et al. (1979) found that plants take up more N in the presence of amoebae and bacteria than with bacteria alone (using the same two species that we used here). Coleman et al. (1977), using the same organisms and similar soil, observed that N mineralization increased with either nematodes or amoebae; but there was no difference between amoebae and nematodes. In all cases, amoebae increased N mineralization; in some cases nematodes did, and in other cases they did not. Nematode populations declined from peak values by the end of their experiment (Coleman et al., 1977); this was also true in our third experiment (Anderson et al., 1981). On the other hand, in our first experiment, the nematode population was at or near its peak (Anderson et al., 1978). We assume that this is also true in our second shorter experiment (14 days). Thus, as long as nematode populations increased or at least maintained high numbers, N mineralization did not increase; but when they declined, N mineralization increased, just as with amoebae. Even though C losses via respiration always increased with nematodes as well as amoebae, N mineralization patterns differed between the two grazers. We calculated the C budget for the first experiment non-glucose-amended treatments (Table 3) (from Coleman et al., 1978). Bacteria contained 669 pg g-l

mated that they contained 1OOpg N g-’ soil since this is a reasonable amount with respect to C and is the amount of total soluble N we extracted from uninoculated soil. Roughly half of this extractable N was NH: (47pg N g-’ soil); the other half was organic N, determined from a Kjeldahl digest of the KC1 extract (Bremner, 1965). (There was less than 1 pg NO;-N g-l soil.) Assuming that bacteria took up 100 pg N g- 1 soil, that amoebae have a C to N ratio of 5 (Reich, 1948), and that nematodes have a C to N ratio of 10 (Meyers and Krusberg, 1965), we calculated a N budget for out first experiment. By further assuming that these organisms excrete no organic nitrogenous wastes, we predicted mineral N concentrations from this budget (Table 4). Because bacterial biomass increased (in bacteria alone treatments) throughout the first experiment (or at least maintained high levels, Anderson et al., 1978) we would not predict any mineralized N. There was none. By the same logic, in our earlier study (ColeTable 3. The distribution of biomass-C (pg g-’ soil) among bacteria, amoebae and nematodes after 24 days in the first experiment

Treatment

Bacteria

B BA BN BAN

669 186 281 218

Biomass-C Amoebae 0 178 0 42

Table 4. The effect of amoebae and nematodes on the calculated distribution of biomass-N and NHf-N (all as /Ig g-’ soil) in the first experiment, after 24 days, assuming 100 pg N in initial bacterial biomass and C to N ratio of 5 for amoebae and 10 for nematodes Treatment

Bacteria

Biomass-N Amoebae

B BA BN BAN

100 28 42 33

0 36 0 8

Nematodes 0 0 1 2

Predicted NH:-N

Observed NH:-N

0 36 57 57

0 32 0 16

Nematodes 0 0 12 16

91

N transformations and microfauna man et al., 1977) we predicted bacteria alone would mineralize N since their biomass first increased, then decreased; presumably taking up N, then releasing it. There were 19 pg mineral N g- ’ soil by the end of the experiment. With amoebae and bacteria together we estimated that there were 28 pg N g- ’ soil in bacteria1 biomass and 36pg N g-’ soil in amoebal biomass after 24 days. Since the bacteria popuIations were not greatly affected in grazed treatments during the first few days (Anderson et al., 19781,we assume that they took up a similar amount of N (100 pg N g- * soil) whether grazed on or not. The expected amount of mineral N with bacteria and amoebae together after 24 days was 36 hg N g- ’ soil; there were 32 pg of mineralized N g -I soil. Continuing the same reasoning, we predicted that nematodes grazing on bacteria would mineralize S?pg N g-’ soil. They did not mineralize any. Like wise, when nematodes fed on both bacteria and amoebae we predicted the same N mineralization (57 pg N g -I soil); these populations mineralized only 16 pg g- ’ soil. If this unaccounted for N were still in nematode biomass its C to N ratio would be unreasonably low (0.2). it seems reasonable to postulate some change in nematode excretory mechanisms when their population declined, as it did in Coleman et aI. (1977)‘s experiment or in our third experiment. When the nematodes exhaust their bacterial food supply, in order to conserve C (now in short supply) they may excrete N (not in short supply) as NH; rather than as organic compounds. Even though respiration increased with nematode grazing (Coleman et al., 19781,and bacteria1 numbers decreased (Anderson er al., 1978), net N mineralization did not necessarily increase. If a change occurred in nematode metabolism, the effects were on N mineralization rather than on community respiration. Effects of trophic interactions in soil microbial populations varied with grazer life history, but the overall trend was clear: The second trophic level tended to increase N mineralization. Further studies have been undertaken to resolve questions about differences in response of grazed and ungrazed bacterial systems to abiotic perturbations, and variations in C and N additions. The results reported here indicate that viewing N mineralization as strictly microfloral is as false for terrestrial as aquatic systems. The different grazers reduced bacterial populations and increased respiration similarly, yet they had different effects on N mineralization. Since these effects vary with the grazers’ life history, it seems that direct excretion by grazers is a more likely cause for enhanced mineralization than purported indirect physiological effects on the bacterial population itself (e.g. Barsdate et al., 1974; Fenchel and Harrison, 1976). Further, since the effect of higher trophic levels was generally to increase mineralization, and since these interactions are so common in soils, we suggest that microfaunal grazing plays an important and direct role in the N cycle of soils equivalent to that suggested for aquatic systems by Johannes (1968). Acknowledgemenrs-We

thank

Michael

Blaha and Jan

Gurnsey for able technical assistance. Support for this research was provided by the Division of Environmental

Biology of the National Science Foundation to Colorado

State University. REFERENCES ANDER~N R. V. and COLEMAND. C. (1981) Population development and interactions between two species of bacteriophagic nematodes. Nematologica 27, 6-19. ANDERSONR. V., ELLIOTTE. T., MCCLELLANJ. F., COLEMAND. C., COLEC. V. and HUNT H. W. (1978) Trophic interactions in soils as they affect energy and nutrient dynamics. III. Biotic interactions of bacteria, amoebae, and nematodes. microbian Ecology 4, 361-371. ANDER~QN R. V., COLEMAND. C., COLE C. V., ELLIOTTE. T. and MCCLELLANJ. F. (1979) The use of soil microcosms in evaluating bacteriophagic nematode responses to other organisms and effects on nutrient cycling. International Journal of Environmental Studies 13, 175-182. ANDERSON R. V., COLEMAND. C., COLEC. V. and ELLIOTT E. T. (1981) Effect of the nemat~~ (Acrobe~oides sp. and ~e~d~~~ogasrer Iheritieri) on substrate utilization and nitrogen and phosphorus mineralization in soil. Ecology 62, 549-555. BARSDATER. J., PRENTKI R. T. and FENCHELT. (1974) Phosphorus cycle of model ecosystems: Significance for deeomposer food chains and effect of bacterial grazers. Oikos 25, 239-251. BREMNERJ. M. (1965) Inorganic forms of nitrogen. In Methods of Soil Analysis, P&t 2, Chemical and Microbiological Properties (C. A. Black. Ed.). tm. 1179-1237. American Society ok Agronomy, Madkoi,‘Wisconsin. CLARK F. E. (1969) Ecological associations among soil microorganisms. In Soil Biology, pp. 125-161. Reviews of Research, UNESCO, Paris. COLEC. V., ELLIOTTE. T., HUNT H. W. and COLEMAND. C. (1978) Trophic interactions in soils as they affect energy and nutrient dynamics. V. Phosphorus transformations. Microbial. E&logy 4, 381-387.. COLEMAND. C., COLEC. V.. ANDERSONR. V.. BLAHAM.. CAMPIONM. K., CLARHOL~M., ELLIOTTE. ?., HUNT fi: W., SCHAEFERB. and SINCLAIRJ. (1977) An analysis of rhizosphere-saprophage interactions in terrestrial econsystems. Ecological Bu~~efjn.Stockholm 25, 299-309. COLEMAN D. C., ANDERSON R. V., COLE C. V., ELLIOTTE. T., WOODSL. E. and CAMPIONM. K. (1978) Trophic interactions as they affect energy and nutrient dynamics. IV. Flows of metabolic and biomass carbon. Microbial Ecology 4,373-380.

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MYERS R. F. and KRUSBERGL. R. (1965) Organic sub-

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OVERGAARDNIELSEN C. (1949) Studies on the soil microfauna-II. The soil-inhabiting nematodes. Natura Jut/andica 2, l-131. REICH K. (1948) Studies on the respiration of an amoeba, Mayorella palestinemis. Physiological Zoolog! 21, 390-412. STANFORD G.. CARTERJ. N.. SIMV~ON E. and SCHWANINGER D. E. (1973) Nitrate determination by a modified Conway microdiffusion method. Journal of the Associurion qf Ojficiul Agricultural Chemists 56, 1365-l 368. YEATESG. W. (1979) Soil nematodes in terrestrial ecosystems. Journal of Nematology 1I, 2 13-228.