Mineralization of nitrogen by protozoan activity in soil

In general, more than 95% of the nitrogen in soils is present in organic forms. This nitrogen is not directly available to plants unless microbial decomposition takes place with the release of mineral nitrogen. In modern agriculture, nitrogen is often applied to arable soils as a fertilizer to support high levels of crop production. Nitrogen is one of the essential nutrients and is required by plants in substantial amounts (1.5-5% x g ^-1dry weight). Extensive application of fertilizer nitrogen causes substantial environmental problems such as leaching of nitrates into groundwater which is used as drinking water and ammonia volatilization into the atmosphere. This ammonia is deposited on the surface of the earth. Nitrification then results in acidification of soils and damage to plants.

The microbial biomass in soil is both a source and a sink for nitrogen. This renders the turnover of nitrogen through the microbial biomass a key process in nitrogen cycling in soil. Knowledge about the mechanisms involved in the mineralization of nitrogen in soils is necessary to improve control of the nitrogen cycling in arable soils. Evidence is accumulating that interactions between microflora and fauna such as protozoa are responsible for a signicant portion of the mineralization of nitrogen in soils. In this thesis, the impact of protozoan predation of bacteria on the mineralization of nitrogen from bacterial cells in soil was investigated.

Traditionally, the mineralization of nitrogen in soils has been attributed to the microflora, i.e. bacteria and fungi. Protozoa have long been recognized as the major predators of bacteria thereby regulating the size of the bacterial populations in soils. The potential impact of protozoa on the mineralization of nitrogen has only recently been recognized (Elliott et al. 1979). When protozoa consume bacteria, excess nitrogen is excreted as ammonium nitrogen. This relation is based on a similar carbon:nitrogen ratio for protozoa and bacteria. Additionally, it has been hypothesized that, by grazing bacteria, protozoa enhance microbial activity and eventually mineralize nitrogen from soil organic matter (Clarholm 1985).

Experiments were carried out in microcosms containing sterilized soil that was inoculated with specific microbial populations. The turnover of the 15 N from microbial cells was assessed by growing plants on these soils and analysing the recovery of 15N in the plant. The activity of protozoa was inferred from an increased number of protozoa during the incubation period.

In Chapter 2, the impact of protozoan grazing on the mineralization of nitrogen from bacterial biomass was investigated. Soil was inoculated with the basic composition of the food chain studied, i.e. ¹⁵N-labelled Pseudomonas aeruginosa alone or with protozoa. Protozoan grazing strongly stimulated the turnover and the mineralization of bacterial nitrogen. In the presence of protozoa, plants recovered 65% more bacterial ¹⁵N than in soils without protozoa. Furthermore, the presence of protozoa resulted in a 26% higher mineralization and uptake of nitrogen from soil organic sources. Additionally, a suspension with a natural bacterial population from the soil was added to the basic composition of the food chain. An increased number of bacteria would immobilize more nitrogen. It was hypothesized that consequently protozoan grazing would exert a more pronounced effect on the mineralization of nitrogen. In this case, the presence of protozoa stimulated the mineralization and uptake by plants of nitrogen from soil organic sources by 44% compared to soils without protozoa. However, the protozoan mediated mineralization and plant uptake of ₁₅ N from bacterial cells was reduced by the addition of the bacterial suspension. This effect was explained by assuming that internal cycling of nitrogen occurred to a larger extent due to an increased size of the bacterial population when both a bacterial suspension and ¹⁵N-labelled Pseudomonas aerughosa were added.

Soil provides a very heterogeneous environment with its network of pores with sizes from 0.2 μm to 2000μm.  Microorganisms have been shown to be neither randomly nor uniformly distributed through the soil fabric (Foster 1988). Part of the habitable pore space for bacteria in soil is not accessible to protozoa (Vargas and Hattori 1986). The numbers of protozoa that are found in natural soil ranges from 1 x 10 ⁴to 1 x 10 ⁶per gram dry soil. Still, protozoa have to migrate through the soil matrix to meet their prey organisms. This is especially true under the experimental conditions in our microcosms where only up to 1 x 10 ⁴protozoa per gram of dry soil have been inoculated at the start. The effect of the inoculum density of protozoa and of spatial separation of protozoa and (prey)organisms on the migration by protozoa as well as on the turnover, dynamics and transport of specific microbial populations in soil was studied (Chapter 3). The CO ₂ evolution served as an indicator of microbial activity. The fate of introduced ¹⁴C-labelled bacterial cells was followed by monitoring ¹⁴C-CO ₂ evolution. Two antibiotic resistant Pseudomonas fluorescens R2f strains were inoculated. The transfer of genes between
representatives of these strains via conjugation was a powerfull indicator of the distribution of bacteria and protozoa in soils. The activity of protozoa increased CO ₂ evolution compared to soils not inoculated with protozoa. This stimulation of the CO ₂ evolution was related to the percentage of a soil portion that was inoculated with protozoa. The inoculated number of protozoa did not affect the CO ₂ evolution. This indicated that protozoa were only active in those particles into which they were inoculated. Protozoa accelerated the turnover of ¹⁴C . The more protozoa were inoculated, the faster specific ¹⁴C-labelled substrates were respired. The turnover of ¹⁴C was determined by the frequency of encounters between protozoa and labelled microorganisms. It was concluded that this frequency is a function of the concentration of protozoa and ¹⁴C-labelled microorganisms in the soil matrix. The results obtained in Chapter 3 picture the soil as a rather constrained system with respect to the mobility of both protozoa and bacteria in the soil matrix. Protozoa hardly influence the distribution of bacterial cells in soil through dispersion. Additionally, it was shown that protozoa did not affect the transfer of genes through organisms in soil. The results support the hypothesis by Stout (1973) that the activity of protozoa is confined to small spaces and consequently small populations.

Protozoa are in essence aquatic organisms and therefore water is essential to their functioning. Based an the average cell sizes of 10-50 μm for amoebae and 10-20 μm for flagellates, these protozoa can only enter and feed in pores with a pore neck diameter 3- 20 μm and larger (Darbyshire 1976). The availability of water was shown to strongly regulate the grazing activity of protozoa (Chapter 5). In soils kept at a low soil moisture tension (3 Bar), protozoa were not active. It was estimated from a water retention curve (Postma et al. 1989) that at this moisture tension, pores with pore necks larger than 3 μm are devoid of water. Hence, protozoan movement and feeding was restricted because waterfilms were too thin or even absent. Only at higher soil moisture contents (0.1 Bar and 0.3 Bar), the activity of protozoa reduced the number of bacteria and increased the mineral nitrogen content in soil (Chapter 5).

Upon drying of the soil, protozoa encyst to survive dry conditions. Little information is available on the signals and the time needed to excyst and return to trophic stages when favourable soil moisture conditions are restored. In a series of three experiments, plant water transpiration was used as an experimental tool to induce soil moisture regimes with defined fluctuations (Chapter 4 and 5). Even though protozoa were forced to encyst upon drying of the soil, they reacted very rapidly to remoistening. In soils that were incubated under conditions with modest soil moisture fluctuations, protozoan activity resulted in an even higher mineralization and plant uptake of ¹⁵N from bacterial cells than in soils that were incubated with a stable soil moisture regime. The activity of protozoa stimulated the mineralization of nitrogen from soil organic sources under all soil moisture regimes applied (Chapter 4). The protozoan activity was further restricted when soil moisture regimes were characterized by more intense and more frequently induced moisture fluctuations (Chapter 5). This was shown by a reduced recovery of bacterial nitrogen in plants compared to soils that were kept continuously moist.

The effect of protozoan predation on the mineralization of carbon and nitrogen from soil organic matter was determined simultaneously in an experiment in which plants were grown in soil microcosms that contained ¹⁴C-carbon and ¹⁵N-nitrogen organic material both in the presence and absence of protozoa (Chapter 6). The predating activity of protozoa accelerated the turnover of microbial carbon and nitrogen by reducing the size of the bacterial population. But more important, protozoa stimulated the activity of the remaining bacterial population as judged from an ongoing higher rate of ¹⁴C-CO ₂ respiration in the presence of protozoa compared to sails without protozoa. Protozoa responded immediately to the restoration of favourable moisture conditions in the soils as shown by the increased rate Of ¹⁴C-CO ₂ respiration. These results support the observations that protozoa numbers increased within 1 or 2 days upon the addition of water to dry soils (Hunt et al. 1989). Furthermore, it was hypothesized that the flow of water contributed to a (re)distribution of protozoa. In soils that were inoculated with less protozoa, the rewetting response in terms of increased rate of ¹⁴C-CO ₂ respiration, was larger than in soils inoculated with more protozoa.

Several mechanisms for the action of protozoa with respect to mineralization of nitrogen have been proposed (Chapter l):
1) by grazing bacteria, protozoan biomass is produced at the expense of bacterial biomass and excess nitrogen is excreted as ammonium
2) by grazing bacteria, protozoa produce waste products (cell wall material and other nutrients) which in turn may enhance microbial activity
3) whilst moving through the soil searching for food particles, protozoa might (re)inoculate (new) substrates by transporting bacteria that adhere to their cell surface or by bacteria that are not digestable and therefore excreted.

In all experiments, the activity of protozoa reduced the size of the bacterial populations both in planted and in unplanted soils by a factor of two (Chapter 5 and 6), five (Chapter 3) to eight (Chapter 2). As a consequence, K is concluded that consumption of bacteria is responsible for at least a part of the increased mineralization of nitrogen in the presence of protozoa. Furthermore, the activity of protozoa induced an ongoing higher microbial respiration rate in planted soils (Chapter 6). It was demonstrated that a smaller sized bacterial population was more active in terms of the amount of carbon metabolized per unit microbial biomass when being predated upon by protozoa. Also, the presence of protozoa increased maintenance of the plasmid in a plasmid containing bacterial population.

This was attributed to an improved nutrient availability in soils where protozoa grazed bacteria. With respect to the third mechanism through which protozoa could enhance mineralization of nitrogen, only limited information was available. It was shown that in unplanted soils (Chapter 3) that are incubated under stable soil moisture regimes, the migration and mobility of protozoa between aggregates is very much restricted (Vargas and Hattori 1986). Based on the observations on gene transfer, it could not be demonstrated that protozoa are important vectors in the allocation of bacteria through soil. However, it was hypothesized that water flow significantly contributes to the distribution of protozoa and probably bacteria as well through the soil matrix.

That microorganisms are responsible for mineralization of nitrogen from organic sources in soils has long been recognized. The results, presented in this thesis, showed clearly that in soils protozoan grazing of bacteria substantially improves the availability of organically bound nitrogen to plants. The mechanisms by which protozoa stimulate the mineralization of nitrogen are i) by releasing nitrogen directly from bacterial cells and ii) by stimulating the turnover of soil organic matter through the microbial biomass. The microcosm approach proved to be useful to study the mineralization of nitrogen by protozoan activity in soil. The dynamic character of planted soil was demonstrated by an ongoing respiration of soil organic carbon. In fallow soil, the protozoan activity lasted only 10 days and then, gradually, microbial activity slowed down. However, in both planted and fallow soil, protozoa exhibited their stimulating effect on carbon and nitrogen mineralization rates. Without significant water flow through the soil matrix, the mobility and migration of protozoa is low. Reallocation of bacteria in the soil matrix was virtually absent. Hence, protozoa sec do not contribute to a (re)distribution and dispersion of bacteria through soil as hypothesized by Finlay and Fenchel (1989). Meanwhile, it was demonstrated that applying a combination of techniques from the field of microbial ecology and of genetics can substantially improve our understanding of the ecology of (introduced) soil microorganisms.

Protozoa exhibited a very fast, immediate reaction to restoration of favourable moisture conditions in previously dried soils. The pulsed addition of water to dry soils have a significant impact on the dynamics of food-chain reactions in soil in terms of microbial activity and of carbon and nitrogen mineralization. Here, the effect of protozoa could be caused both by substrates made available to microorganisms through disruption of soil aggregates or through an improved distribution of the biota through the soil. The magnitude of the effect of protozoan grazing on the plant availability of organically and bacterial bound nitrogen supports the concept that food-web interactions rather than microbial activity alone, are responsible for nitrogen mineralization in soil.