Antonie van Leeuwenhoek 81: 309–318, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Bacteria and protozoa as integral components of the forest ecosystem –their role in creating a naturally varied soil fertility

Marianne ClarholmSwedish University of Agricultural Sciences, Department of Forest Mycology and Pathology, Box 7026, SE-750 07

Uppsala, Sweden (E-mail: Marianne.Clarholm@mykopat.slu.se)

Key words: ciliates, flagellates, microbial loop, N mineralization, naked amoebae

Abstract

The paper explores interactions between the two first organism groups to appear on earth, the bacteria and protozoa,and their interplay with the rest of the ecosystem focusing upon northern boreal forests. The microbial loop issuggested as a mechanism for local inputs of new N to the ecosystem. The possibility to couple short-term microbialprocesses with their long-term effects, – as registered in plants, soil and the atmosphere, via the abiotic variables –is explored. The latter are investigated in relation to the environments they create for the micro-organisms, and howthis results in varying soil fertility. A chain of events is presented that relate high Ca concentration in the mineralsoil and high water availability to increased nitrogen availability for plants via the micro-organisms. An exampleis given of the influence of these parameters directly upon protozoa along an extreme fertility gradient, and alsoindirect evidence from a Finnish field study of 30 sites with four fertility levels. Finally, there is a discussion aboutways to convert knowledge gained in detailed studies of microbial interactions into forms useful when evaluatingthe present status of and effects of ameliorative management on ecosystems strongly affected by humans.

Introduction

Bacteria and protozoa have thus far been more in-vestigated in agricultural ecosystems than in forests.One reason could be that man-made food-producingecosystems have generally been more in focus for re-search than naturally established extensively managedforests. Nonetheless in recent years there have beenmany reports of alarming changes in some forest eco-systems, both in Europe and in the USA that generallycan be connected to strongly increasing human in-fluences. Field observations of substantial changes inforest trees and in soils, but also the possibility to storemore C in boreal forest soils, have raised our interestin the functioning of forested ecosystems (Janssens etal. 2001). It has become obvious that ‘the actual forcesstructuring natural ecosystems are not yet understood’(Tilman 1998). This lack of knowledge has causedproblems when interpreting our results and in our at-tempts to understand the seriousness of the changeswe observe. It also diminishes our potential to coun-

teract the observed changes optimally or to choosebetter alternatives than simply attempting to return theecosystem to the state where it was before.

Most of the range of biological processes re-ported to react to global changes are performed bymicro-organisms. Today the nature of environmentallimitations of soil microbial activities is less clearlyunderstood than the limiting factors for plants (Vance& Chapin 2001). Soil microbiologists therefore have aspecial responsibility to contribute new knowledge inthis area. Doing so, we have to handle both the recog-nized dichotomy between studies of micro-organismsand studies of microbial processes, and the linkagesbetween the invisible microbiology and the visibleparts of the ecosystem. If we are to unravel the pro-cesses and parameters that shape ecosystems, a logicalfirst approach is to study naturally established sys-tems as unaffected as possible by human activities.In Scandinavia there are still forests that broadly fulfilthese prerequisites.

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Soil fertility: the tree connection

The most conspicuous organisms in forested ecosys-tems are the trees. Most of our quantitative knowledgeis focused on tree productivity because of its eco-nomical importance. Scientifically there is uncertaintyabout the mechanisms creating varying productivitylevels. Different empirical ways to classify the poten-tial productivity of a forest soil have developed withindifferent groups working with forests.

Foresters need a sturdy field method to judge sitequality and they use indicator plants, mostly fromthe field layer. Soil scientists use inherent soil char-acteristics. They have empirically demonstrated thatthe productivity of forest soils increases with increas-ing concentrations of calcium and magnesium in themineral layer, especially in sites that also have anample water supply. The latter is judged from theplant species cover and from the leveling of the sur-face. Concentrations of Ca and Mg in the upper 10 cmof the mineral soil as well as cation exchange capa-city have been found to correlate positively with treegrowth (Alban 1974; Shrivastava 1980; Jokela et al.1988). Soil biologists use the carbon/nitrogen (C/N)ratio and the pH of the organic layer where more fertilesites have lower C/N and higher pH than less fertilesites. Possibly there is an influence due to the fact thatN and C are fixed in biological processes, and N isthe only nutrient that will immediately increase treegrowth when added to forests on mineral soils.

The methods of the forester, the soil scientist andthe soil biologist all three work to predict soil fertil-ity, is it possible, therefore, that they relate to eachother? One approach in the study of soil fertility isto work to unravel the mechanisms that lie behind thevarying amounts of N available for trees at locationswith differing natural productivity. At first thought,the available N should be the difference between ad-ditions and losses from a common pool available totrees, that is to say the same scenario as in the man-made agricultural situation characterized by a surplusof N, a low C availability for the micro-organisms anda pH just below neutral. Scandinavian forests consistmostly of conifers with a mor humus forest floor whereN is growth-limiting, the potential C availability ishigh relative to that of N, they have a pH between4 and 5 and the absolute majority of the finest rootsare covered with mycorrhiza-forming fungi. The N ac-quisition is more complex than in agricultural soils andfor the moment incompletely understood. It is likelythat the N supply mechanisms differ between more

and less fertile soils. In Scandinavia two main treespecies, Norway spruce (Picea abies Karst) and Scotspine (Pinus sylvestris L.), cover a range of fertilitylevels and will adjust their growth to the local circum-stances. There are never visible symptoms of nutrientimbalances in natural forest situations uninfluenced byman, just more or less growth increment.

N flows to and from forest ecosystems of varying

productivity

The biological transfer of N between the large atmo-spheric pool and the soil is the exclusive responsibilityof bacteria. Input occurs through N-fixation. Denitri-fication and N2O formation in connection with nitrific-ation exports N back to the atmosphere. Judged from atime series, individual boreal forest soils seem to accu-mulate both C (Liski & Westman 1997) and N (J Liski,pers. comm.) up to a plateau value. This would implythat once this value is reached, in the studied case afteraround 1200 years, gaseous losses and leaching fromthe soil would roughly equal the inputs as fixation byfree-living bacteria, deposition and the accumulationin live vegetation.

N fixation and denitrification are processes that areextremely patchy both in space and time. The methodsused to estimate the fluxes could easily be criticized(Binkley et al. 2000). If symbiontic N fixing trees,mainly alder, are disregarded, there have been fewquantitative measurements of N-fixation performed inforest ecosystems and even fewer of denitrification.The few estimates available indicate an increasingflow of N through the ecosystem with increasing fertil-ity. For a productive mixed deciduous forest in SouthCarolina, USA, where both biological N-fixation anddenitrification were measured, values of 10–18 kgha−1 year−1 were reported for both processes (Todd etal. 1978). Amounts of N fixed in mixed fairly product-ive forests dominated by conifers in central Swedenwere estimated to 1–2 kg ha−1 year−1, in an areawith a moderate N deposition of around 8 kg ha−1

year−1. The fixation was concentrated to the top cmand increased strongly with increasing pH and withthe presence of birch (Nohrstedt 1985). A more north-ern low productive not fully closed 120-year-old pineforest on poor sandy soil had a measurable N fixationof around 0.3 kg ha−1 year−1 associated with the fieldlayer and the roots (Granhall & Lindberg 1978). Thisvalue should be compared with an N deposition of 5kg year−1.

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What are the external prerequisites for N fixationand denitrification (Table 1)? Some obligate prerequis-ites vary between processes. N fixation requires theabsence of available N, while denitrification requiresthe presence of N as NO3

− or NO2−. The processes

also have common prerequisites. They both need localanaerobic conditions around the performing bacteriaand a local pH of 4.5 or higher (Weber & Sundman1986). An energy source, generally C, is involvedin both processes. N fixation is always energy de-manding, but a C source also promotes denitrification,indirectly through consumption of O2, creating an-aerobicity, and directly as a C source. Carbon is anecessary prerequisite for growth of N-fixing hetero-trophic bacteria, once the N is fixed. The C qualityis crucial. Bacteria are generally only able to utilizerather simple molecules, which are rapidly digested.

Plant litter quality and the C/N of the forest floor

The availability for micro-organisms of C in plant lit-ter depends upon the chemical composition of the C.The phenol content of plants is used as an indicatorof substrate quality and it is affected by the nutrientavailability. Both soluble and total amounts of phenolsin the forest floor increase with decreased soil fertility(Muller et al. 1987; Northup et al. 1995). Fertilizationexperiments have shown that the same tree species hasthe lowest phenol levels under high (sufficient) nutri-ent availability, and that phenol levels will increasewhen N or P availability is low (Davies et al. 1964).As regards cations, a positive relation have been re-ported between degree of base saturation in a soil andthe carbohydrate content of litter from pines grown atthe same site (Sanger et al. 1998). There are also in-terspecific differences, with conifers generally havinghigher levels of phenols than deciduous trees under thesame nutrient conditions. High phenol content makeslitter more resistant to decomposition. Carbon boundin phenols will be largely unavailable for bacteria.

All kinds of litters contain around the same amountof C (≈50%) which can be of varying quality (Berget al. 1999). Needles and roots (Muller et al. 1989)grown under fertile conditions will contain less phen-ols and thus larger amounts of more easily availableC sources than the same plant parts grown under lessfertile conditions. Parts of the non-phenol C will bea suitable bacterial substrate and more C will there-fore be available for N fixation in litter with lowphenol content. Increased N fixation rates have been

recorded in litter of the same tree species with increas-ing substrate quality measured as decreasing ligninconcentration (Hobbie 2000).

Another mechanism that makes substrate lessavailable for micro-organisms is when OH-groups onthe phenols react with amino acids, e.g., from mi-crobial remains, and form stable protein–phenol com-plexes. This will make both the N and C in the proteinunavailable for bacteria. This reaction is suggested tobe one cause of the lower N availability in soils withlow natural fertility (Hättenschwiler & Vitousek 2000)and also to the build up of large stores of virtually in-ert organic N in agricultural soils (Nömmik & Vahtras1982). In organic matter-based agriculture, substratequality, measured as phenol content, has been recog-nized as an important variable in the timing of nutrientdelivery (Palm & Sanchez 1991). Phenols seem to beconnected to nutrient delivery also in natural systems,possibly with more than one mechanism involved.

The declining C/N of the forest floor connectedwith increasing soil fertility is most likely is causedby a comparatively fast decomposition rate of the Cfraction, but also by an absolute increase of N throughN fixation and a lock-up of N in protein–phenolcomplexes.

Carbon metabolisation related to Ca

concentration

C quality and C availability are difficult to measurein litter and soils so a multi-method approach can behelpful. A C substrate of high quality will be metabol-ised at a higher rate than a substrate of lower quality,but material of the same substrate quality will alsobe more easily decomposed in patches with higherpH. Both situations result in increased CO2 evolutionrate. In a spruce forest with mixed pines, a strongcorrelation was found between Ca concentration andbasal respiration in the forest floor for 34 samplestaken in a grid with 10 m between samples (Palmborget al. 1998). The respiration rates showed a 3 folddifference and the Ca concentrations varied 4 foldamong the sampled cores, demonstrating a consid-erable small-scale variation. The results indicate apositive relation between an increased base cation con-tent (in turn transforming into increased pH) and anincreased rate of C substrate utilization. A high basecation content has also been empirically connected tohigh soil fertility (Jokela et al. 1988; Lundell 1989).Hesselman (1926) reported that the same plant species

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Table 1. Prerequisites for nitrogen fixation and denitrification

Variable Status required for N Status required for

fixation denitrification

Inorganic N Absent NO3− present

Oxygen concentration Low Low

pH Minimum ≈4.5 Minimum ≈4.5

Temperature Lower limit 5 ◦C Lower limit 5 ◦C

P requirements High Unknown

C requirements An available C source A C source will promote

is needed for energy for N anaerobicity and growth

fixation and for growth under anaerobic conditions.

grown under more fertile soil conditions containedmore base cations. Higher CO2 evolution rates indic-ate higher microbial metabolic rates, and most likelyit also implies microbial growth.

Water as a transporter

Water, the second factor found to promote productiv-ity is affecting an ecosystem in more than one way. Itfunctions as a nutrient in its own right, participating inthe metabolic processes in all live organisms. It formswater films for micro-organisms and roots to be meta-bolically active within, and it also acts as a mechanismof transport. Ions are transported downwards in thesoil profile by passive water flow and upwards throughevaporation, within fungal hyphae and tree roots. Wa-ter moves cations from all parts of the soil profile intothe trees, and onto the surface as plant litter.

Soil moisture and water transport within soils aredifficult to measure and will vary much with time andspace. In models, water transport through trees is afunction of precipitation, temperature and evapotran-spiration. Plant physiologists use the Ca content ofcurrent year’s needles in the autumn as a first crudeestimate of the water used in photosynthesis duringthe past growing season. A high Ca content indic-ates a high water flow through the tree. The transportcapacity of water for Ca was demonstrated in an irriga-tion experiment with Norway spruce in south-westernSweden, where the yearly uptake of Ca increasedby 20% from 20 to 25 kg ha−1 year−1 when waterdeficiencies were reduced (Nilsson & Wiklund 1994).

The Ca–water–soil fertility connection

The question is now: How does the amount of cations(mainly Ca2+ and Mg2+) transported by water throughthe tree, and subsequently deposited on the surfacein litter influence the N availability for trees? Thelikely mechanism is that increasing Ca concentrationsform local environments around and inside surfacelitter and root litter that are more favorable for bothN mineralisation and N fixation than the situation atlarge. Bacterial enzymes are more effective in lessacid environments and an increased pH will increaserelease rates of C and N. Litter with sufficiently highpH (>4.5) will allow local N fixation when the restof the prerequisites (Table 1), are fulfilled, building Ninto bacterial biomass and in time dead bacterial andprotozoan remains. The latter will be easily decom-posed and will contribute to higher N availability forplants in cation rich soils. Good nutrient availability inturn results in less phenol-rich plant litter with high Cavailability, decomposability and a higher pH than therest of the forest floor, creating a positive feed-backloop.

Protozoa quickly release nutrients in bacteria

When the small amount of inorganic N generallypresent in fresh litter is consumed, the situation inthis microhabitat will locally become suitable for Nfixation. Through growth, nutrients will be enrichedwithin bacteria. When bacteria die through consump-tion or due to adverse conditions, nutrients will bereleased locally in higher concentrations than earlier.Other organisms, e.g., mycorrhizal fungi, which ob-tain C directly from the trees, could thereafter take

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up and circulate the nutrients. The N could be trans-ferred to the tree partner. However, isotope studieshave revealed that a N molecule will circulate manytimes between various members of the soil flora beforeit leaves the ground and enters a plant (Kaye & Hart1997).

A pulse of bacterial production created throughgrowth on a limited C input from fresh plant litteror newly dead roots, is production over the ‘carryingcapacity’ of a site and will be short-lived, since it im-mediately triggers the growth of bacteria-feeding pro-tozoa (Clarholm 1981). A bacterial production pulsecan therefore easily be overlooked (Clarholm 1994).Protozoa are the most effective of all bacterial feedersbecause of their feeding and growth habit; they eat firstand divide later. Protozoa will quickly develop a highgrazing pressure since they multiply through divisionwith potentially as short generation times as bacteria.They are small (4–150 µm diameter), plastic and lacka proper cell wall when in an active state. Protozoathus have easy access to the small water filled spacesand surfaces where bacteria grow (Bamforth 1987).

And then they die?

The fate of protozoa is not clear. In the field they de-crease from peak numbers within days (Cutler et al.1922; Clarholm 1981, 1989). The average numbersrecorded suggest that the majority of the individuals ina peak do not form cysts, but die in one way or other.Protozoa live at least partly in spaces to which lar-ger possible predators theoretically have little access.In models protozoa are often consumed by predatorynematodes, but also by earth-worms and enchytraids.However, such feeding activities have actually neverbeen observed (Zwart et al. 1994). An alternate possib-ility to predation is that the majority of the individualsfail to encyst and just die when food bacteria becomeabruptly scarce, or when the soil dries out. An indica-tion of this behaviour is the fact that protozoa cysts areresistant in animal guts and an individual protozoonthat is ingested as a cyst should still be viable after gutpassage and show up in a MPN determination.

With the interactions described, two-thirds of abacterial production (and newly fixed N) will becomedead organic C and N in just 5 days (Clarholm 1981),while one-third has been converted to CO2 and am-monium (Figure 1). The organic remains of bacteriaand protozoa will be difficult to distinguish from therest of the older organic material, but will still consti-

tute a suitable, high quality food for micro-organismsand detritial feeders. For earthworms in fertile soils,which actually seem to be dependant on the presenceof protozoa for normal growth (Miles 1963), for en-chytraeids (Didden 1993) and testate amoebae in theleast fertile soils. In agricultural soils newly formedorganic N is recognized as a far more reactive andaccessible N source than older organic N (Paul 1984).Presumably the same holds for natural soils. Micro-organisms are in both cases the most obvious sourceof this new organic N.

Time series and their microbiology

In early soil forming processes, the pH of the freshbedrock is around neutral. In sites carrying borealforests, periodically high precipitation and leachingwill decrease pH with time, and more so at lessproductive locations as compared to more product-ive ones. The balance between the cations addedby weathering and lost permanently by leaching, ortemporarily through plant uptake, will reach an equi-librium creating the level of acidity in today’s borealforest soils. For an individual site, a coniferous forestwith a mor type forest floor seems to be a stablesituation, which can persist for thousands of years ac-cording to pollen analysis, but it could be broken andredirected by fire. Fires initiated by climate change,turning into a situation with more dry spells, and thusless leaching, will lead to a situation where the con-ifers are substituted for deciduous trees (Willis et al.1997).

Studies of the microbiology of age-gradientsformed on land when glaciers withdraw have also beenconducted (Ohtonen et al. 1999). Virgin land sur-faces with neutral pH became colonised, first with amicro-flora mainly dominated by bacteria and lichenswith associated N-fixing blue–green bacteria, there-after plants with symbiontic N-fixing bacteria fol-lowed. Here the main source for N to the plants wasobviously the atmosphere. With increasing age anddecreasing pH non-fixing tree species took over, andfungi increased their presence in the forest floors inthe studied gradients, while the bacterial compartmentremained of the same size.

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Figure 1. A schematic diagram of the microbial loop, showing how the major part of pulse bacterial production is transformed to various formsof dead organic matter within five days. Only the small part that ends up as protozoan cysts is still alive.

Field observations at 30 Finnish sites with varying

productivity

Are there any field observations to support the ideaspresented on how varying soil fertility is created andmaintained? Not directly performed with this purpose,but extensive Finnish investigations of C storage andturnover can be examined in this respect. In southernFinland, an area with low N deposition, 30 conifer-ous forest sites with varying productivity levels and allwithin 200 km of each other, have been investigatedby soil scientists (Liski & Westman 1995; Liski et al.1997) and soil microbiologists (Pennanen et al. 1999).

The sites were divided into four fertility classesbased on indicator plants in the field layer. The twoleast productive classes the Calluna type (CT) and theVaccinium vitis-idea type (VT) carried pines, whilethe two most productive the Myrtillus type (MT) andOxalis Myrtillus type (OMT) carried spruce trees.Base saturation in the FH layer increased from 19 to45% with increasing fertility. Base cations originatefrom the mineral layers, and the fertile site classes hadthe largest stores of base cations in the E and C hori-zons, and the sites with the weakest productivity had

the lowest concentrations. The distribution of cationswith higher concentrations in the forest floor than inmineral layers did indicate a transportation of Ca frommineral layers to the surface via the trees.

Water availability could be indirectly judged fromthe level of the ground water surface and the soil’scapacity to conserve water, which increases with anincreasing silt fraction. The ground water level wassimilar for both pine classes, and lower comparedwith the spruce classes. The concentration of water-conserving silt was lower in CT than in the otherthree classes. It is likely that the ground water wasthe highest in the most productive site class, because itseemed to have a similar or lower silt fraction than MT.Unfortunately no ground water value was reported forthe OMT sites.

The C in the standing crop of trees more thandoubled from the lowest to the highest productivity,while the differences were smaller for the C stored insoil. Since more litter is produced in absolute terms inmore productive sites, the latter indicates that the litterin the more productive sites had been of better qualityand its C content had been mineralised more rapidly bythe micro-organisms. The reported observations sup-

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port an increased cation transport from more cationrich mineral layers onto the organic forest floor in themore productive treatments. The transport was gener-ated by a higher water flow through trees, resultingin more Ca ending up in the needles. This will in turnlead to fresh litter with a locally increased pH, improv-ing enzyme performances, decomposition rates andnutrient availability, in turn improving the C qualityof the litter and the circumstances for N-fixation.

When microbial variables were measured on oneoccasion in September there were no differences inthe mixed humus layer neither in basal respiration,for total amounts of micro-organisms, or for bacteriawhen estimated with phospholipid fatty acid analysis(Pennanen et al. 1999). The only difference foundwas for fatty acids associated with fungi, where therewas a significant decrease with increasing productiv-ity. The same standing crop of micro-organisms, butwith varying composition of the microbial communityhad contributed to the different amounts of C foundin trees and soil. The higher absolute amounts of Clost from the soil at the sites with the more productiveforest types also indicate that more microbial N hadbeen turned over. When the whole humus layer wasassayed as one sample, the pH differences betweenfertility classes were significant, but the absolute rangewas small (4.0–4.2). This could possibly indicate thatincreased water availability allowing translocation ofnutrients within the trees, was the prime cause ofhigher production at these sites. Another possibilityis that only a small upper part of the humus layer hadan increased pH (see Table 2), and by mixing this partwas diluted with a much larger part with lower pH.

A second example: the fertility gradient in Betsele

In northern Sweden, 150 km from costal Umeå in alow N-deposition area (2–5 kg ha−1 year−1) a steepnutrient gradient has been localised with a range inpH of more than 2 units in the forest floor across adistance of only 90 m. The gradient is naturally de-veloped after a forest fire 160 years ago and has beendescribed chemically and botanically (Giesler et al.1998). At the low pH end it starts with an area withpine as the dominant tree species with a dwarf-shrubunder-storey. Thereafter follows a short-herb area witha mixture of pines and spruce trees, ending with atall-herb area with coarse spruce trees and in the veryend, which also is a water discharge area, a glade withfallen spruce trees. The height index (height at 100

Table 2. Naked amoebae, flagellates and ciliates [no g−1 dw × 10−3] with depth in the forest floor at 20 m and 40 m in the Betselegradient (M. Clarholm, unpublished)

Layer 20 m 40 m 20 m 40 m

Naked amoebae pH

L1 379 3988 4.57 6.79

L2 374 1559 4.49 6.66

F 7 968 4.35 6.09

H 6 719 3.92 5.15

Flagellates

L1 329 6270

L2 450 1515

F 202 1291

H 120 960

Ciliates Dry weight/Fresh weight

L1 12 29 0.42 0.27

L2 23 29 0.31 0.26

F 3 4 0.30 0.26

H 2 3 0.35 0.23

years) spans from 17 to 28 m with a basal area varyingfrom 22 to 32 m2 ha−1. The tree growth indicates nat-urally varying site fertility. There is a strong variationin base saturation (18–70%), N concentration (1.2–3.2%) and C/N ratios (45–18) in the forest floor alongthe gradient (Högberg et al. 1990).

Protozoa along the Betsele gradient

The most recently fallen surface litter was sampledalong the Betsele gradient in September 2000 for es-timation of protozoa. This is the compartment that isexpected to vary most in substrate quality (Melillo etal. 1989) and also to be a favourable place for bac-terial growth. From 80 m onwards there were onlysparse quantities of surface litter to sample. Thereforelive material/soil made up part of the samples, whichthus were not directly comparable with the rest of thegradient.

Very large populations of naked amoebae (Fig-ure 2), one hundred times larger than in the rest ofthe gradient, were recorded in the samples from 0,10 and 20 m where recently fallen pine needles werethe major component that was almost lacking in therest of the samples. Scots pines shed around 70% oftheir needle litter over a short period concentrated inthe autumn contributing fresh C. The favourable effectof fresh pine litter on numbers of naked amoebae has

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Figure 2. Naked amoebae, flagellates and ciliates (no. g dw−1) inlitter along a fertility gradient in Betsele in northern Sweden in mid-September 2000. The lowest fertility at 0 m, for more details see thetext (M. Clarholm, unpublished).

been observed before when the whole thin forest floorwas sampled, also that time in September (Clarholm1981). Naked amoebae need a substantial bacterialproduction to multiply, indicating that fresh pine lit-ter constitutes a good but finite C source for bacteria.Thanks to the great plasticity of the amoebal cell, thebacteria can serve as food within the couple of µm thinwater films in the dry pine part of the gradient.

From the 40-m sample with a pH(H2O) of 5.5and 20 m onwards, ciliates became an increasinglyprominent part of the protozoa (Figure 2). Their com-paratively high number is an indication of a substantialbacterial production, but especially a good water avail-ability, at least periodically, since members of the mostsoil-adapted ciliate group, the Colpoda, need a waterfilm of at least 30 µm to be active (Fenchel 1994). Anincreasing bacterial production should be coupled toan increasingly improved availability of C for bacteria.At 70 m the bacterially-available litter C presumablywas of even higher quality than at 60 m and most ofit already consumed at the time of sampling and thebacteria-consuming ciliates were likewise turned over.Flagellates followed the same general pattern as cili-ates, suggesting bacteria as a common food source (M.Clarholm, unpublished).

A preliminary study of the protozoa of the L, Fand H layers in the forest floor was conducted withsamples from 20 and 40 m. The largest numbers ofnaked amoebae, flagellates and ciliates were foundin the top layer (L1) at both distances. The pH(H2O)

followed the same pattern (Table 2). The decrease in

numbers within the forest floor was more distinct at20 m as compared to 40 m, indicating a higher bac-terial production that is to say better C availabilityfurther down in the forest floor at 40 m. The watercontent (Table 2) at 40 m was substantially higher thanthat at 20 m, connecting increased water availabilitywith increasing soil fertility.

These concomitant measurements in two mainforest types that normally cover large areas on theirown, but here exist close together under the same cli-mate regime, show that processes connected to the Nturnover are not synchronized in time between foresttypes. Instead the bacterial production and possible in-puts of new N are most likely connected to the inputof new C, which will vary in time with plant species.

The evolution and impact of man

An evolutionary perspective can help us to understandthe role of bacteria and protozoa in relation to therest of the ecosystem. Bacteria were first to appearon earth, protozoa appeared second. Their activitieslaid the foundation for the rest of the terrestrial bio-sphere by introducing nutrients via weathering andfixation (Bacteria) and increasing the rate of nutrientturnover (Protozoa). But what determines the activitiesof the bacteria? The Dutch microbiologist Baas Beck-ing wrote about bacteria in a book on environmentalmicrobiology ‘Everything is everywhere, the environ-ment decides’ (Baas Becking 1934). To understand,and possibly counteract, unwanted bacterial activit-ies and favour wanted ones, we thus should focusmore on the environment where the bacteria are act-ive and try to understand how environmental changeshave altered the prerequisites for their activities. Arecent example is the increased inputs of airborne N-containing pollutants by man substituting for N-fixingbacteria, which have created situations never experi-enced before by micro-organisms, like acid forest soilswith large amounts of inorganic nitrogen. Without hu-mans, more neutral soil conditions are needed to createenvironments rich in mineral N. But it was artificiallycreated and soon nitrifiers capable of performing dur-ing the new more acid conditions also evolved (DeBoer et al. 1992), allowing excess nitrogen to leavethe ecosystem.

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How to connect micro-organisms, microbial

processes, and their ecosystem effects via abiotic

parameters

Microbiologists have stressed the difficulty of estab-lishing the link between micro-organisms and theprocesses they perform. So when is this a necessaryprerequisite for solving the questions we ask? Mostmicrobiological processes are performed by differ-ent micro-organisms depending on the local environ-mental conditions, and the important information iswhether the process is performed or not, and whatare the driving forces that affect the locally perform-ing micro-organisms. The effects of the driving forceson the studied process should be worked out at themechanistic level. This will require studies with sub-stantial detail and focused hypotheses. In studies of aprocess like N-mineralization in forest soils, the crudeparameter ‘microbial biomass’ has not led us forward(Bauhus & Khanna 1999). Most likely because, de-pending on the circumstances, the microbial C andN biomass is locked up in different micro-organismsthat behave differently and react to different drivingforces, but perform the same process, N mineralization(Vance & Chapin 2001).

Once the basic biology is known for a central mi-crobial process in soil, the next step is to performsystematic investigations to establish the relationsbetween the micro-organisms, the process studied andthe driving variables under a spectrum of typical soilconditions in places with low human influence. Anindividual site will be classified as disturbed whencentral parameter values, identified in the detailed in-vestigations above, diverge a certain degree from whathave been recorded for the same type of site in anundisturbed situation. With knowledge about micro-bial processes and their driving forces, we will alsohave a situation where we can discuss the merits anddrawbacks of possible amelioration initiatives.

Based on empirical studies, C/N ratio in the topsoil, with values varying depending on tree species,are proposed for example for practical use to predictN leaching following clear-cutting (Dise et al. 1998).Possibly the local pH in fresh litter together with Caconcentration in the mineral layer of a site could yieldinformation on the N availability of an ecosystemin the future, once the proposed relations have beenfurther evaluated.

Conclusions

The individual level of productivity of a local forestsite is suggested to depend on the mineralogy of thesoil, particularly its content of Ca and Mg, and thewater availability for the vegetation. The absoluteamounts of cations in fresh litter will influence ratesof mineralization of organic N, N fixation and thusN availability. This will in turn affect the C qualityof new needles and their suitability as a C source forbacterial N fixation, creating a feed-back loop.

Bacterial protozoan interactions should be studiedin detail to understand their role in bringing new Ninto the ecosystem, a process that substitutes inevit-able N losses and determines the absolute levels of Navailability.

When microbiological processes are considered atecosystem level, soil micro-organisms should not beinvestigated directly once their environmental drivingforces are known. With that knowledge it should bepossible to classify an individual site and evaluate itspresent situation by the degree of deviation of the stud-ied parameters as compared to values from an equiv-alent site under undisturbed conditions. It should alsobe possible to forecast how intentional (fertilization)and unintentional (diffuse pollution) human additionswill affect the performance of the micro-organisms.

Acknowledgements

I would like to thank Ulf Rubinsson for permittingaccess to his land in Betsele. Constructive commentson the MS from B. Heal, H. Setälä, P.A. Wookey, B.Lindahl and I. Fernandez were most helpful.

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