Microbial ureases: significance, regulation, and molecular characterization.

Microbial functioning refers to microbial activity because only the active microorganisms drive biogeochemical processes. Despite the importance of active microorganisms, most methods focus on estimating total microbial biomass and fail to evaluate its active fraction. At first, we have described the differences among the active , potentially active , and dormant microbial states in soil and suggested threshold values of parameters for their identification. Secondly, we critically reviewed the ability of a broad range of approaches to estimate and characterize the active and the potentially active microorganisms in soil. Following approaches were evaluated: plate count and microbial cultures; direct microscopy combined with cell staining; ATP, PLFA, DNA and RNA content; microarray analyses; PCR-based approaches; stable isotope probing; soil proteomics, enzymes activity; and various approaches based on respiration and substrate utilization. The “static” approaches, mainly based on the single-stage determination of cell components (ATP, DNA, RNA, and molecular biomarkers), detect well the presence of microorganisms and total biomass, but they fail to evaluate the active part and consequently the functions. In contrast, the dynamic approaches, estimating the changes of these parameters during microbial growth and based on process rates: substrate utilization and product formation, e.g., respiration, help to evaluate active microbial biomass and relate it to specific process rates. Based on a comparison of all approaches for their universality (possibility to analyze active, potentially active and dormant microorganisms), we concluded that 1) direct microscopy with complementary stains, 2) a combination of RNA-based FISH with staining of total microbial biomass, and 3) approaches based on microbial growth were the most advantageous and allowed simultaneous quantitative estimation of active , potentially active, and dormant microorganisms in soil. The active microorganisms compose only about 0.1–2% of the total microbial biomass and very seldom exceed 5% in soils without input of easily available substrates. Nonetheless, the fraction of potentially active microorganisms (ready to start utilization of available substrates within few hours) is much higher, contributing between 10 and 40% (up to 60%) of the total microbial biomass. Therefore, we emphasize the role of potentially active microorganisms with quick response to fluctuating substrate input in soil microhabitats and hotspots. The transition from the potentially active to the active state occurs in minutes to hours, but the shift from dormant to active state takes anywhere from hours to days. Despite very fast activation, the reverse process – fading to the potentially active and dormant stage – requires a much longer period and is very different for individual criteria: ATP, DNA, RNA, enzyme production, respiration rates. This leads to further difficulties in the estimation of the active part of microbial community by methods based on these parameters. Consequently, the standardization, further elaboration, and broad application of approaches focused on the portion of active microorganisms in soil and their functions are urgently needed. We conclude that because active microorganisms are the solely microbial drivers of main biogeochemical processes, analyses of the active and potentially active fractions are necessary in studies focused on soil functions.

Active microorganisms in soil: Critical review of estimation criteria and approaches

A long-term field experiment utilising barley received four different treatments prior to sowing: municipal solid waste (MSW) compost at either 20 t ha 21 (C20) or 80 t ha 21 (C80); cow manure (MA) at 20 t ha 21 ; mineral fertilizer (MIN) or NPK (400 kg ha 21 ); and NH4NO3 (150 kg ha 21 ). The effects of these applications on soil enzyme activities and microbial biomass at crop harvest were measured after nine years. In comparison with the control (no amendment) MSW addition increased biomass C by 10 and 46% at application rates of 20 and 80 t ha 21 , respectively, while MA treatment increased microbial biomass C by 29%. The ratio of soil microbial C to soil organic C was the lowest at the high rate of MSW application. Oxidoreductase enzymes, such as dehydrogenase and catalase, were higher in the MSW treatments by 730 (C20) and 200% (C80), respectively, and by 993 and 140% in MA treatments than in the unamended soil, indicating an increase in the microbial metabolism in the soil as a result of the mineralization of biodegradable C fractions contained in the amendments. The addition of MSW and MA caused different responses in hydrolase enzymes. Phosphatase activity decreased with MSW (^62% at both rates) and MA (^73%), to less than those in the mineral fertilization and the control treatments. Urease activity decreased by 21% (C20) and 28% (C80), possibly being affected by the heavy metals contained in the MSW. However,b-glucosidase and protease-BAA increased in all the organic treatments, especially with MA (by 214 and 177%, respectively). This is attributed to the microbial stimulation by the organic C and is correlated with the increase in dehydrogenase Or 2 a 0:882U and catalaseOr 2 a 0:654U activities. q 2000 Elsevier Science Ltd. All

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Long-term effects of municipal solid waste compost application on soil enzyme activities and microbial biomass

Nitrification, a microbial process, is a key component and integral part of the nitrogen (N) cycle. Soil N is in a constant state of flux, moving and changing chemical forms. During nitrification, a relatively immobile N-form (NH 4 +) is converted into highly mobile nitrate-N (NO 3 −). The nitrate formed is susceptible to losses via leaching and conversion to gaseous forms via denitrification. Often less than 30% of the applied N fertilizer is recovered in intensive agricultural systems, largely due to losses associated with and following nitrification. Nitrogen-use efficiency (NUE) is defined as the biomass produced per unit of assimilated N and is a conservative function in most biological systems. A better alternative is to define NUE as the dry matter produced per unit N applied and strive for improvements in agronomic yields through N recovery. Suppressing nitrification along with its associated N losses is potentially a key part in any strategy to improve N recovery and agronomic NUE. In many mature...

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Scope and Strategies for Regulation of Nitrification in Agricultural Systems—Challenges and Opportunities

(1976). Extracellular Enzymes in Soil. CRC Critical Reviews in Microbiology: Vol. 4, No. 4, pp. 383-421.

Extracellular Enzymes in Soil

The effects of washings of plant roots on the rate of nitrification in soil was investigated. The root washings were obtained by leaching columns of quartz chips in which plants were growing, with their roots bathed in nutrient solution. The root washings and ammonium solution were then added to columns of clay loam soil in which ammonium-ion was being converted to nitrate-ion at the rate of c. 370 mg N/kg soil/day. Rates of nitrate formation and ammonium disappearance were estimated by analysis of leachates from the soil columns. The root washings of all plants investigated (ryegrass, wheat, salad rape, lettuce and onion) reduced the rate of nitrification but the effects of rape and lettuce were temporary. Ryegrass root washings had the most pronounced and persistent effects and reduced the rate of nitrification (up to 84 per cent) in proportion to concentration. The rate of nitrification was not limited by supplies of ammonium-ion or other nutrients and there was no reduction of nitrate in the soil columns. As neither microbial immobilization of inorganic-nitrogen nor denitrification appeared to be taking place in the presence of root washings, it was concluded that root washings can in some way retard nitrification although no mechanism was demonstrated.

The influence of washings of living roots on nitrification

In a laboratory investigation of the volatilization of ammonia following the application of urea to an acid loamy sand it was found that the loss of ammonia could be delayed for several days and the maximum rate of loss could be reduced by the addition to the soil of various monohydroxamic acids. All the hydroxamic acids used are non-competitive inhibitors of the enzyme urease but in the soil system caprylo-hydroxamic acid and phenylaceto-hydroxamic acid were ineffective in retarding ammonia loss and on a molar basis acetohydroxamic acid (AHA) was a more effective inhibitor than either benzo-, capro- or salicylohydroxamic acid. The effectiveness of AHA in delaying ammonia loss was proportional to its concentration at any one concentration of urea. AHA added to soil several days before the application of urea did not delay losses of volatile ammonia. This may have been due either to chemical inactivation or biological decomposition of AHA or to the proliferation of ureaseforming organisms tolerant towards AHA. When either sulphanilamide, copper sulphate or sodium diethyldithiocarbamate were added to the soil they caused slight delays in ammonia loss but they were far less effective than AHA. The delay caused by AHA treatment was prolonged in the presence of sulphanilamide and this effect was attributed to toxic effects of sulphanilamide on urease-forming organisms.

The influence of hydroxamates on ammonia loss from an acid loamy sand treated with urea

A series of laboratory experiments were made to investigate the influence of seven monohydroxamic acids on the loss of ammonia from soils that had been treated with urea. The hydroxamic acids (aceto-, propiono-, capro-, caprylo-, benzo-, salicylo- and phenylaceto-hydroxamic) are all non-competitive inhibitors of the enzyme urease. Acetohydroxamate (AHA) was tested in 16 different soils and it reduced the maximum rate of ammonia loss from 3 sands, 4 loamy sands and 3 sandy loams and in most of them it delayed the onset of ammonia loss. The effects of AHA on ammonia evolution from 6 soils with high clay contents were variable and although ammonia losses were small from these soils AHA brought about an increase in the rate of loss from two of them. Sulphanilamide was a less effective inhibitor than AHA but when both inhibitors were applied together to an acid sandy loam they brought about a prolonged delay of ammonia volatilization but they were not synergistic when applied to a neutral sandy loam. Sodium diethyldithiocarbamate and benzohydroxamate both delayed losses from a calcareous clay loam but five of the hydroxamates accelerated the rate of ammonia volatilization. Copper sulphate did not appear to delay ammonia loss but reduced the total loss from the calcareous clay loam and also from a sandy loam.

The influence of hydroxamates on ammonia loss from various soils treated with urea

Autoradiographic procedures are described to detect metabolically-active hyphae in litter habitats. Experiments with Sclerotium rolfsii and with hyphae present on the surfaces of leaf litter of beech showed that active hyphae could be detected after exposure for 2 hr to 10 μCi/ml of uniformly-labelled 14C-glucose followed by film exposure for 2 days. Controls indicated that there was negligible background radioactivity and that there was no adsorption or non-metabolic (passive) uptake of radionuclide by hyphae. Liquid photographic emulsion was superior to stripping film because closer contact could be obtained between the hyphae and the emulsion. Use of photographic emulsion was facilitated by removing hyphae from leaf litter surfaces in polystyrene peels and then covering the polystyrene peels with emulsion. The use of these techniques in other habitats in soil is discussed.

J.S. Waid

Papers

The influence of washings of living roots on nitrification

The influence of hydroxamates on ammonia loss from an acid loamy sand treated with urea

The influence of hydroxamates on ammonia loss from various soils treated with urea

A method to detect metabolically-active microorganisms in leaf litter habitats