Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h^-1. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h^-1, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10⁵ and 12 × 10⁵ flagellates ml^-1 were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h^-1, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

[This corrects the article on p. 1964 in vol. 58.].     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Soil Columns

The enhanced mineralization of immobilized nitrogen by bacteriophagous protozoa has been thought to favor the nitrification process in soils in which nitrifying bacteria must compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the flagellate Adriamonas peritocrescens on the competition for ammonium between the chemolithotrophic species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi was studied in soil columns, which were continuously percolated with media containing 5 mM ammonium and different amounts of glucose at a dilution rate of 0.007 h^-1 (liquid volumes). A. globiformis won the competition for ammonium. The grazing activities of the flagellates had two prominent effects on the competition between N. europaea and A. globiformis. First, the distribution of ammonium over the profile of the soil columns was more uniform in the presence of flagellates than in their absence. In the absence of flagellates, relatively high amounts of ammonium accumulated in the upper layer (0 to 3 cm), whereas in the underlying layers the ammonium concentrations were low. In the presence of flagellates, however, considerable amounts of ammonium were found in the lower layers, whereas less ammonium accumulated in the upper layer. Second, the potential ammonium-oxidizing activity of N. europaea was stimulated in the presence of flagellates. The numbers of N. europaea at different glucose concentrations in the presence of flagellates were comparable to those in the absence of protozoa. However, in the presence of flagellates, the potential ammonium-oxidizing activities were four to five times greater than those in the absence of protozoa.     

Competition between Picoplanktonic Cyanobacteria and Heterotrophic Bacteria along Crossed Gradients of Glucose and Phosphate

A laboratory experiment was performed to test whether differences in nutrient and energy demands between picophytoplankton and heterotrophic bacteria can explain the apparent inverse biomass relationship between these organisms in lakes along gradients of organic carbon and nutrients. Growth rates and final yield of cells were analyzed in crossed gradients of glucose and phosphate. Concentrations of phosphate (10, 25, and 60 microg P L(-1)) and glucose (0, 0.3, and 3 mg C L(-1)) were used in all possible combinations giving 9 different treatments. Heterotrophic bacteria had higher maximum growth rates in all treatments and became larger than picophytoplankton in many treatments. The variance in abundance of heterotrophic bacteria between treatments could almost completely be explained by the combined effects of glucose and P. In treatments where carbon limitation slowed down the growth of heterotrophic bacteria, picophytoplankton became abundant and these organisms showed a positive response to P in combination with a negative response to glucose. The negative effect of glucose on picophytoplankton is suggested to be indirect and caused by competition with bacteria that are favored by organic C. The results suggest that competition for phosphate between phytoplankton and bacteria is not size-dependent, that heterotrophic bacteria are superior competitors for P, and that organic C produced by picophytoplankton was of minor importance for heterotrophic bacteria.     

异养硝化-好氧反硝化菌脱氮相关酶系及其编码基因的研究进展

水体氮素污染日益严重,如何经济、高效地去除水体氮素已成为研究热点。近年来,研究人员已从不同环境中分离到许多同时具有异养硝化和好氧反硝化功能的菌株,此类菌生长迅速,可在好氧条件下同时实现硝化和反硝化的过程,并可用于脱除有机污染物,是一类应用潜力巨大的脱氮菌。目前,异养硝化-好氧反硝化菌的脱氮途径和机制主要是通过测定氮循环中间产物或终产物、测定相关酶活性、注释部分氮循环相关基因及参考自养硝化菌和缺氧反硝化菌的氮循环途径等进行研究,其完整的氮素转化途径和氮代谢机制还需要进一步明确。总结了目前异养硝化-好养反硝化菌的脱氮相关酶系及其编码基因的研究进展,以期为异养硝化-好氧反硝化菌的理论研究及其在污水脱氮处理上的应用提供参考。     

Nitrifying Bacteria in Wastewater Reservoirs

Deep wastewater reservoirs are used throughout Israel to store domestic wastewater effluents for summer irrigation. These effluents contain high concentrations of ammonia (≤5 mM) that are frequently toxic to photosynthetic microorganisms and that lead to development of anoxic conditions. Population dynamics of nitrifying bacteria and rates of nitrification were studied in two wastewater reservoirs that differed in organic load and degree of oxygenation and in the laboratory under controlled conditions, both by serial dilutions in mineral medium and microscopically with fluorescein isothiocyanate-conjugated antibodies prepared against local isolates. The difference in counts by the two methods was within 1 order of magnitude. In the laboratory, an O₂ concentration of 0.2 mg liter⁻¹ was close to optimal with respect to growth of NH₃ oxidizers on domestic wastewater, while O₂ concentrations of 0.05 mg liter⁻¹ supported significant rates of nitrification. It was found that even hypertrophic anaerobic environments such as the anaerobic hypolimnion of the wastewater reservoir or the anaerobic settling ponds are capable of sustaining a viable, although not actively nitrifying, population of Nitrosomonas spp. and Nitrobacter spp., in contrast to their rapid decline when maintained anaerobically in mineral medium in the laboratory. Nitrification rates of NH₃ in effluents during storage in the reservoirs were slower by 1 to 2 orders of magnitude compared with corresponding rates in water samples brought to the laboratory. The factors causing this inhibition were not identified.     

Effects of pH and Oxygen and Ammonium Concentrations on the Community Structure of Nitrifying Bacteria from Wastewater

Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter⁻¹), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant. The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined. Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis. Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level. The smaller changes in community structure brought about by the two pH extremes, however, were irreversible. Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative, Nitrosomonas eutropha C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the β subgroup of the class Proteobacteria. A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles.Autotrophic nitrifying bacteria that oxidize ammonium to nitrite and nitrate are found in soils, sediments, wastewaters, freshwater, and marine water and on building facades. They are essential components of the nitrogen (N) cycle, linking the most reduced and most oxidized forms of inorganic N. Nitrification occurs as a two-step process carried out by two distinct groups of bacteria; ammonia-oxidizing bacteria convert ammonia to nitrite, and then nitrite oxidizers convert nitrite to nitrate (22, 30). Environmental factors control the rate of nitrification. The most significant environmental factors are substrate concentration, pH, temperature, and oxygen availability (12, 23). Nitrifying bacteria exhibit different substrate concentration sensitivities (26). Media containing low substrate concentrations (10 mg of NH₄⁺ liter⁻¹) can give larger most-probable-number counts of ammonia oxidizers than media containing higher NH₄⁺ concentrations (6, 26). Also, ammonia oxidation is inhibited at high substrate concentrations. The growth rates of Nitrosomonas spp. cultures were reduced in the presence of 1,050 to 2,800 mg of NH₄⁺-N liter⁻¹ (16). Substrate inhibition of ammonia oxidation has also been observed in studies of wastewater systems (23). Natural environments, such as soil and water, usually contain 1 to 10 mg of NH₄⁺-N liter⁻¹ (22), yet liquid wastes from animal farms give rise to concentrations up to 1,600 or 5,600 mg of NH₄⁺-N liter⁻¹ (5, 17). Free ammonia (NH₃) rather than the total ammonium concentration inhibits ammonia oxidizers (1). As the ratio between the ionized form and the nonionized form depends on pH, the toxicity of ammonium also depends on the environmental pH.The pH range for growth of pure cultures of ammonia oxidizers is 5.8 to 8.5, and the pH range for growth of nitrite oxidizers is 6.5 to 8.5 (30). Nitrification was inhibited at pH values below 5.8 in our preliminary experiments performed with an enriched culture of nitrifiers obtained from wastewater. Yet in natural environments, such as soil, nitrification has been reported to occur at pH values below 4.0 (7, 29).Limiting amounts of dissolved oxygen (concentrations below 2 mg liter⁻¹) inhibit nitrification and cause nitrite accumulation or nitrous and nitric oxide production (9, 21). Ammonia-oxidizing bacteria are the key functional group in removing ammonium from wastewaters. Knowledge of the effect of oxygen on nitrification and nitrifying populations has economic importance since aeration of activated sludge is one of the most costly items in the operation of a wastewater treatment plant (21).In environments with high inputs of ammonium, such as wastewaters, biooxidation of this substrate increases the oxygen uptake and lowers the pH. Such modifications of the environment not only affect the production of nitrite and nitrate but can also select a different nitrifying community that is perhaps specialized for these new conditions. Nitrification does occur in extreme environments that pure cultures of nitrifiers cannot tolerate (4). In this study we examined extreme environments in which nitrifying bacteria may be viable but have not been cultured thus far.Because of the difficulty of obtaining nitrifier isolates, nucleic acid-based methods have greatly aided studies of the diversity of nitrifiers (11, 20, 27, 28). Recent molecular investigations have provided valuable information concerning the diversity of ammonia oxidizers in natural environments (5, 15, 20, 25). However, no previous study has focused on the structural or compositional responses of nitrifying communities to perturbations in the environment. In the present laboratory study we examined the effects of high ammonium concentrations, different pH values, and different oxygen concentrations on nitrification and on the community structure of nitrifying bacteria from wastewater. To test the abilities of the communities to regain their original structures, growth of nitrifying communities under the new conditions was followed by incubation under the original conditions.     

Competition of Octopine-Catabolizing Pseudomonas spp. and Octopine-Type Agrobacterium tumefaciens for Octopine in Chemostats

The ability of two octopine-catabolizing Pseudomonas spp. and two virulent octopine-type Agrobacterium tumefaciens to compete for substrates has been examined in chemostats. In dual cultures with octopine or glutamate as the limiting carbon or nitrogen source, Pseudomonas fluorescens B99A and E175D always dominated over A. tumefaciens B6 or ATCC 15955. The growth dynamics of each strain in pure culture indicated that some form of antagonism was occurring in dual culture to permit the predominance of the pseudomonads under certain conditions. Although both pseudomonads fluoresce, pyoverdine was not responsible for the observed inhibition. An unidentified antibiotic secreted by both pseudomonads is believed to be responsible. A. tumefaciens B6 grew synergistically in the presence of P. fluorescens B99A with octopine as the limiting nitrogen source. This behavior of Agrobacterium strain B6 may help overcome its grossly inefficient use of octopine as previously reported. The ability of these two pseudomonads to outcompete the agrobacteria under all conditions tested raises the possibility that under field conditions, infectious agrobacteria may be succeeded by opine-catabolizing pseudomonads around crown gall tumors and in the rhizosphere.     

Weak Coupling between Heterotrophic Nanoflagellates and Bacteria in a Eutrophic Freshwater Environment

In a study on the dynamics and trophic role of the heterotrophic nanoflagellate (HNAN) assemblage in the microbial food web of a eutrophic oxbow lake abundances, biomass, and production rates of HNAN and their potential prey organisms, namely heterotrophic bacteria and autotrophic picoplankton, were monitored for a period of 2 years. No coupling between HNAN abundance and biomass and the abundance and biomass of their picoplanktonic prey was observed for the investigation period. The ratio of heterotrophic bacterial to HNAN abundance ranged from 2.2 x 103 to 8.6 x 103 (mean: 4.2 x 103 +/- 1.8 x 103). HNAN carbon consumption could account for only 10% to 40% of bacterial secondary production. The lack of coupling between HNAN and their potential prey and the low HNAN abundance relative to bacterial abundance suggested (a) that HNAN grazing was an insignificant factor in the regulation of bacterial abundance and (b) that HNAN abundance was regulated by predation rather than by prey abundance. This hypothesis was supported by the fact that HNAN growth rates were high (in the range of 0.45 d-1 to 1.00 d-1 during spring and summer, yearly mean: 0.52 d-1), and only weakly correlated with prey abundance and biomass. The results indicated strong top-down control of HNAN and consequently a weak coupling of HNAN and picoplankton in the investigated eutrophic freshwater environment.     

Nitrification and Autotrophic Nitrifying Bacteria in a Hydrocarbon-Polluted Soil

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.     

Competition between Transposable Elements and Mutator Genes in Bacteria

Although both genotypes with elevated mutation rate (mutators) and mobilization of insertion sequence (IS) elements have substantial impact on genome diversification, their potential interactions are unknown. Moreover, the evolutionary forces driving gradual accumulation of these elements are unclear: Do these elements spread in an initially transposon-free bacterial genome as they enable rapid adaptive evolution? To address these issues, we inserted an active IS1 element into a reduced Escherichia coli genome devoid of all other mobile DNA. Evolutionary laboratory experiments revealed that IS elements increase mutational supply and occasionally generate variants with especially large phenotypic effects. However, their impact on adaptive evolution is small compared with mismatch repair mutator alleles, and hence, the latter impede the spread of IS-carrying strains. Given their ubiquity in natural populations, such mutator alleles could limit early phase of IS element evolution in a new bacterial host. More generally, our work demonstrates the existence of an evolutionary conflict between mutation-promoting mechanisms.     

Ammonium Toxicity in Bacteria

Although an excellent nitrogen source for most bacteria, ammonium was—in analogy to plant and animal systems—assumed be detrimental to bacteria when present in high concentrations. In this study, we examined the effect of molar ammonium concentrations on different model bacteria, namely, Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The studied bacteria are highly resistant to ammonium. When growth was impaired upon addition of molar (NH₄)₂SO₄ concentrations, this was not caused by an ammonium-specific effect but was due to an enhanced osmolarity or increased ionic strength of the medium. Therefore, it was concluded that ammonium is not detrimental to C. glutamicum and other bacteria even when present in molar concentrations.     

Grazing, Growth, and Ammonium Excretion Rates of a Heterotrophic Microflagellate Fed with Four Species of Bacteria

We studied aspects of the population growth of a microflagellate, Monas sp., isolated from Lake Kinneret, Israel. The protozoan growth rates, rates of ingestion of bacteria, and final population yields generally increased with increasing bacterial concentrations, although the exact relationship varied depending on the species of bacteria used as food. Grazing rates decreased hyperbolically with increasing food density. Gross growth efficiencies and ammonia excretion rates were similar over a range of food densities among the four species of bacteria. Population doubling times and ammonia excretion rates were lowest, and growth efficiencies were highest, at temperatures between 18 and 24°C. Under optimum conditions, the microflagellates had average population doubling times of 5.0 to 7.8 h, average growth efficiencies of 23.7 to 48.7%, and average ammonia excretion rates of 0.76 to 1.23 μmol of NH₄⁺ per mg (dry wt) per h.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Distribution of Heterotrophic Bacteria in Lake Shira

A study of the horizontal and vertical distribution of heterotrophic bacteria in brackish Lake Shira in summer periods showed that mesophilic bacteria dominated in all areas of the lake, whereas psychrotolerant bacteria dominated in the metalimnion and hypolimnion of its central part. Nonhalophilic bacteria were mostly mesophilic and dominated in coastal waters. Most psychrotolerant bacteria were able to grow in the presence of 5–10% NaCl. Heterotrophic bacteria isolated in different regions of the lake were identified to a generic level. The isolates were classified into autochthonous and allochthonous microorganisms on the bases of their distribution pattern in the lake water, halotolerance, and ability to grow at low temperatures.     

Length of Incubation for Enumerating Nitrifying Bacteria Present in Various Environments

The effect of incubation time on most-probable-number estimates of autotrophic nitrifying bacteria was investigated by using waters, rooted aquatic plants, sediments, and slimes as inoculum sources. Maximum most probable numbers of the NH₄⁺-oxidizing group were attained in 20 to 55 days (median, 25). Estimates of NO₂^- oxidizers were highest at termination (103 to 113) days.     

Mechanisms for Soil Moisture Effects on Activity of Nitrifying Bacteria

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.