Formic Hydrogenlyase and the Photoassimilation of Formate by a Strain of Rhodopseudomonas palustris

A photosynthetic bacterium isolated by enrichment on media containing formate as major source of cell carbon was identified as a strain of Rhodopseudomonas palustris. It grew on a wide range of simple organic compounds including alcohols, fatty acids, and hydroxyacids, on a chemically defined medium with biotin and p-aminobenzoic acid as essential growth factors. The organism grew on formate or photoautotrophically with molecular hydrogen or thiosulfate only in the presence of yeast extract. Ability to photoassimilate formate could be shown only in organisms grown in the presence of formate. The organism contained an inducible formic hydrogenlyase consisting of a soluble formic dehydrogenase, a particulate hydrogenase, and one or more intermediate, but as yet unidentified, electron carriers. The formic hydrogenlyase could be reconstituted from a particulate hydrogenase and a partially purified soluble formic dehydrogenase. Some properties of the formic dehydrogenase and hydrogenase have been compared with that of the formic hydrogenlyase system.     

Induced Biosynthesis of Formic Hydrogenlyase in Iron-Deficient Cells of Escherichia coli.

Fukuyama, T. (University of Washington, Seattle), and E. J. Ordal. Induced biosynthesis of formic hydrogenlyase in iron-deficient cells of Escherichia coli. J. Bacteriol. 90:673-680. 1965.-Escherichia coli cells were grown aerobically on a lactate-mineral salts medium from which iron had been removed by extraction with 8-hydroxyquinoline and chloroform. These cells carried out induced biosynthesis of formic hydrogenlyase in a reaction mixture containing glucose, formate, and phosphate without the addition of amino acids, providing adequate amounts of iron salts were present. In the absence of iron, glucose was fermented and acids were produced, but no formic hydrogenlyase developed. When iron-deficient E. coli cells were repeatedly washed, the property of carrying out induced biosynthesis of formic hydrogenlyase with glucose, formate, phosphate, and iron was lost, but was restored on addition of acid-hydrolyzed casein to the reaction mixture. An energy source (provided as glucose) was necessary for enzyme production. Iron-deficient cells were devoid of hydrogenase and formic hydrogenlyase but showed formic dehydrogenase activity when adequate amounts of selenium and molybdenum were present in the growth medium. Hydrogenase was consistently absent in iron-deficient cells but appeared concomitantly with formic hydrogenlyase during induced biosynthesis of the latter in iron-deficient cells of E. coli.     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

Structural and Functional Features of Formate Hydrogen Lyase, an Enzyme of Mixed-Acid Fermentation from Escherichia coli

Formate hydrogen lyase from Escherichia coli is a membrane-bound complex that oxidizes formic acid to carbon dioxide and molecular hydrogen. Under anaerobic growth conditions and fermentation of sugars (glucose), it exists in two forms. One form is constituted by formate dehydrogenase H and hydrogenase 3, and the other one is the same formate dehydrogenase and hydrogenase 4; the presence of small protein subunits, carriers of electrons, is also probable. Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel-cobalt). Its formation also depends on the external pH and the presence of formate. Activity of both forms requires F(0)F(1)-ATPase; this explains dependence of the complex functioning on proton-motive force. It is also possible that the formate hydrogen lyase complex will exhibit its own proton-translocating function.     

Coenzymes of methanogenesis from hydrogen and carbon dioxide

Methanogenic bacteria gain their energy for growth from the conversion of a number of simple carbon compounds to methane. With a few exceptions all species known to date are able to reduce CO₂ at which hydrogen acts as the electron donor. The reduction of CO₂ can formally be considered to proceed through the formyl, the formaldehyde and the methyl level of reduction. These C₁-units do not occur as free intermediates, but they remain bound to a number of unique coenzymes during the process. In this paper a survey is given of the structures and functions of these compounds; it deals with methanopterin derivatives, carbon dioxide reduction (CDR) factor, factor F₄₃₀ and coenzyme M derivatives. A model of the process of methanogenesis that integrates previous ones and that allocates a function to the various coenzymes is presented.This paper is adapted from a treatise by the same author, entitled Coenzymes of methanogenesis. that was awarded the Kluyver prize 1984 by the Netherlands Society for Microbiology.     

Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly--hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO₂ and H₂ were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H₂. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.Abbreviations G+C guanine plus cytosine - OD optical density - PHB poly--hydroxybutyric acid - specific growth rate - HPLC high-performance liquid chromatography - YE yeast extract     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Secretion to the growth medium of an α-amylase by Escherichia coli clones carrying a Bacillus laterosporus gene

-Amylase gene from Bacillus laterosporus P₃ was cloned and expressed in Escherichia coli HB101 and DH5. Up to 92% of the cloned gene product was secreted into the medium by the recombinant E. coli. The recombinant crude enzyme showed improved functionality in terms of activity at a wider pH range and at higher temperature, as compared to the crude enzyme from the donor strain. The improved functionality of the cloned enzyme was due to the absence of a contaminating protease which was co-produced in the donor strain. Sub-cloning of the -amylase gene using the promoter-probe vector, pKT240 in E. coli DH5 indicated the presence of a promoter of B. laterosporus P₃ in the cloned fragment.     

Expression of fungal desaturase genes in cultured mammalian cells

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and a-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, 12 and 15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal 12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo--linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of 12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal 6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.     

Reductive Pentose Cycle and Formate Assimilation in Rhodopseudomonas palustris

Rhodopseudomonas palustris assimilated formate autotrophically as carbon dioxide and hydrogen arising from the activity of the formic hydrogenlyase system. Kinetic analyses of cell suspensions pulse-labeled with (14)C-formate or (14)C-bicarbonate showed similar distributions of incorporated radioactivity. In both cases phosphate esters were the first assimilation products. Ribulose diphosphate carboxylase, phosphoribose isomerase, and phosphoribulokinase, characteristic enzymes of the reductive pentose cycle, were present in extracts of cells grown on formate.     

Anaerobic degradation of phenylacetate and 4-hydroxyphenylacetate by denitrifying bacteria

From various oxic or anoxic habitats anaerobic enrichment cultures were set up which completely oxidized aromatic amino acids to CO₂ with nitrate as electron acceptor. Tyrosine and tryptophan at first were degraded to phenol and indole, respectively, prior to utilization of the aromatic ring; with phenylalanine no intermediates were detected. Attempts to isolate denitrifying bacteria able to completely degrade aromatic amino acids were unsuccessful. Starting with these enrichments several strains of denitrifying bacteria were anaerobically enriched and isolated with known fermentation products of amino acids (phenylacetate, 4-OH-phenylacetate, 2-OH-benzoate) plus nitrate as sole sources of carbon and energy.Three strains were characterized further. They grew well in defined mineral salts medium, were gram-negative and facultatively anaerobic with strictly oxidative metabolism; molecular oxygen, nitrate or nitrite served as electron acceptors. The isolates were tentatively identified as pseudomonads, but could not be aligned to known species. They oxidized a variety of aromatic compounds completely to CO₂ anaerobically and, with some exceptions, also aerobically. The substrates included among others: (4-OH)-phenylacetate, (4-OH)-phenylglyoxylate, benzoate, 2-aminobenzoate, phenol, OH-benzoates, indole and notably toluene. Reduced alicyclic compounds were not utilized. During anaerobic degradation of (4-OH)-phenylacetate transient accumulation of (4-OH)-phenylglyoxylate was observed.It is proposed that anaerobic -oxidation of the-CH₂–COOH side chain to -CO–COOH initiates anaerobic degradation of (4-OH)-phenylacetate. This implies a novel type of anaerobic -hydroxylation with water as the oxygen donor. Abbreviation. Hydroxyl groups were abbreviated as OH     

Assessment of <Emphasis Type="Italic">Nannochloropsis gaditana</Emphasis> growth and lipid accumulation with increased inorganic carbon delivery

Algal biomass refineries for sustainable transportation fuels, in particular biodiesel, will benefit from algal strain enhancements to improve biomass and lipid productivity. Specifically, the supply of inorganic carbon to microalgal cultures represents an area of great interest due to the potential for improved growth of microalgae and the possibility for incorporation with CO₂ mitigation processes. Combinations of bicarbonate (HCO₃^?) salt addition and application of CO₂ to control pH have shown compelling increases in growth rate and lipid productivity of fresh water algae. Here, focus was placed on the marine organism, Nannochloropsis gaditana, to investigate growth and lipid accumulation under various strategies of enhanced inorganic carbon supply. Three gas application strategies were investigated: continuous sparging of atmospheric air, continuous sparging of 5% CO₂ during light hours until nitrogen depletion, and continuous sparging of atmospheric air supplemented with 5% CO₂ for pH control between 8.0 and 8.3. These gas sparging schemes were combined with addition of low concentrations (5 mM) of sodium bicarbonate at inoculation and high concentration (50 mM) of sodium bicarbonate amendments just prior to nitrogen depletion. The optimum scenario observed for growth of N. gaditana under these inorganic carbon conditions was controlling pH with 5% CO₂ on demand, which increased both growth rate and lipid accumulation. Fatty acid methyl esters were primarily comprised of C16:0 (palmitic) and C16:1 (palmitoleic) aliphatic chains. Additionally, the use of high concentration (50 mM) of bicarbonate amendments further improved lipid content (up to 48.6%) under nitrogen deplete conditions when paired with pH-controlled strategies.     

Reaction engineering analysis of hydrogenotrophic production of acetic acid by Acetobacterium woodii

Great interest has emerged in biological CO₂‐fixing processes in the context of current climate change discussions. One example for such a process is the hydrogenotrophic production of acetic acid by anaerobic microorganisms. Acetogenic microorganisms make use of carbon dioxide in the presence of hydrogen to produce acetic acid and biomass. In order to establish a process for the hydrogenotrophic production of acetic acid, the formation of acetate by Acetobacterium woodii was studied in a batch‐operated stirred‐tank bioreactor at different hydrogen partial pressures (pH₂) in the gas phase. The volumetric productivity of the batch processes increased with increasing hydrogen partial pressure. A maximum of the volumetric productivity of 7.4 g_acetate L⁻¹ day⁻¹ was measured at a pH₂ of 1,700 mbar. At this pH₂ a final acetate concentration of 44 g L⁻¹ was measured after a process time of 11 days, if the pH was controlled at pH 7.0 (average cell density of 1.1 g L⁻¹ cell dry weight). The maximum cell specific actetate productivity was 6.9 g_acetate g day⁻¹ under hydrogenotrophic conditions. Biotechnol. Bioeng. 2011;108: 470–474. © 2010 Wiley Periodicals, Inc.     

Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H₂ evolution due to the formate hydrogenlyase reaction. H₂ production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO₂ production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H₂ was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.     

A generalized model of a resource-population system

Summary A system of nonlinear differential equations describing a resource-population system is analyzed in terms of the existence and characteristics of its equilibrium states.It is proved that, under the condition that k< (necessary condition for the population being able to grow under optimal conditions), it is a necessary and sufficient condition for the system to have a steady state that the resource input rate to the system be constant.When the resource input rate is a constant different from zero, the system has only one equilibrium point, at M ⁰=/⁰/k, A ⁰=–(/⁰/k)ln(1–k/), and this equilibrium point is always stable. In other words, the system population-resource will always reach the steady state, either monotonically (node) or by damped oscillations (focus), from any arbitrary initial condition in the positive quadrant.When the resource input rate is equal to zero, the system has an infinite number of equilibrium points at M ₀=0, A ₀=constant. All these equilibrium points are unstable in the sense that any slight increase in M will move the system away from the equilibrium states, except for the point M ₀=0, A ₀=0, which is the only stable equilibrium point, to which the system will tend. This stable equilibrium point corresponds to the condition of complete annihilation of both resource and population.Finally, it is proved that the system does not have limit cycles in the positive quadrant and is therefore incapable of self-oscillations.This work was partially supported by a Ford Foundation fellowship and various Cornell University fellowships.     

The role of oxygen in the regulation of the metabolism of aerotolerant spirochetes,a major component of <Emphasis Type="Italic">“Thiodendron”</Emphasis> bacterial sulfur mats

Two spirochete strains isolated earlier from Thiodendron bacterial sulfur mats grew better under microaerobic (0.3–0.5 mg O₂/l) than under anaerobic conditions. The microaerobic growth of these strains was accompanied by a twofold increase in the cell yield and the efficiency of glucose utilization, despite the fact that an additional amount of ATP (and, hence, glucose) was spent in this case for the synthesis of exopolysaccharides. Glucose metabolism under microaerobic conditions gave rise to more oxidized products (acetate and carbon dioxide) than under anaerobic conditions (formate, ethanol, pyruvate, and hydrogen). The paper considers two putative mechanisms implemented by aerotolerant spirochetes: adaptive (the use of a more efficient pathway of glucose catabolism) and protective (an enhanced synthesis of exopolysaccharides and the reduction of hydrogen peroxide by the reduced sulfur compounds thiosulfate and sulfide, yielding elemental sulfur). The formation of Thiodendron bacterial sulfur mats in saltwater environments is also discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 725–733.Original Russian Text Copyright © 2004 by Dubinina, Grabovich, Chernyshova.     

Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells

Production of a TGF receptor with high density baculovirus infected Sf-9 cells (7×10⁶cells ml^-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGF receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO₂ partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO₂ at this concentration has been assessed at smaller scale for TGF receptor and -gal expression, in controlled atmosphere incubators.Abbreviations CST cumulative sparging time (s) - dpi days post-infection (d) - CO₂/O₂ diffusivities ratio (=0.784 at 27 °C) - DO % saturation of dissolved oxygen (%) - hpi hours post-infection (h) - He Henry coefficient (He_{CO 2}=32.1×10^-3M atm^-1, He_{O 2}=1.23×10^-3 M atm^-1 at 27 °C) - k_La volumetric liquid-side mass transfer coefficient (h^-1) - LDH Lactate dehydrogenase - MOI multiplicity of infection (pfu cell^-1) - N agitation speed (rpm) - OTR oxygen transfer rate (mole O₂ ml^-1 h^-1) - OUR oxygen uptake rate (mole O₂ ml^-1 h^-1) - p partial pressure (atm) - P total pressure (atm) - pfu plaque forming units - q specific consumption or production rate (mole cell^-1 h^-1) - Q_{H, OUT} headspace outlet gas flow rate - Q_S,NOM nominal volumetric sparged gas flow rate (92 ml s^-1 at bioreactor conditions) - R ideal gas constant (82.05 ml atm mole^-1 K^-1) - SR_{V L} molar sparging rate per unit liquid volume (mole ml^-1 h^-1) - SSR specific sparging rate (mole cell^-1 h^-1) - T temperature (C or K) - V_L culture volume (ml) - VVD volume of feed per volume of culture per day (d^-1) - X cell concentration (cell ml^-1) - Y yield coefficient Indexes CO₂, O₂ related to CO₂ or O₂, respectively - glc glucose - H headspace gas phase or gas/liquid interface - L liquid phase - lact lactate - S sparged phase or gas/liquid interface - V viable     

Bioconversion of carbon dioxide to methane using hydrogen and hydrogenotrophic methanogens

Biogas produced from organic wastes contains energetically usable methane and unavoidable amount of carbon dioxide. The exploitation of whole biogas energy is locally limited and utilization of the natural gas transport system requires CO₂ removal or its conversion to methane. The biological conversion of CO₂ and hydrogen to methane is well known reaction without the demand of high pressure and temperature and is carried out by hydrogenotrophic methanogens. Reducing equivalents to the biotransformation of carbon dioxide from biogas or other resources to biomethane can be supplied by external hydrogen. Discontinuous electricity production from wind and solar energy combined with fluctuating utilization cause serious storage problems that can be solved by power-to-gas strategy representing the production of storable hydrogen via the electrolysis of water. The possibility of subsequent repowering of the energy of hydrogen to the easily utilizable and transportable form is a biological conversion with CO₂ to biomethane. Biomethanization of CO₂ can take place directly in anaerobic digesters fed with organic substrates or in separate bioreactors. The major bottleneck in the process is gas-liquid mass transfer of H₂ and the method of the effective input of hydrogen into the system. There are many studies with different bioreactors arrangements and a way of enrichment of hydrogenotrophic methanogens, but the system still has to be optimized for a higher efficiency. The aim of the paper is to gather and critically assess the state of a research and experience from laboratory, pilot and operational applications of carbon dioxide bioconversion and highlight further perspective fields of research.     

Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C

Ten strains representing a novel genus of marine thermophilic archaebacteria growing at between 70 and 103°C with an optimal growth temperature of 100°C and a doubling time of only 37 min were isolated from geothermally heated marine sediments at the beach of Porto di Levante, Vulcano, Italy. The organisms are spherical-shaped, 0.8 to 2.5 m in width and exhibit monopolar polytrichous flagellation. They are strictly anaerobic heterotrophs, growing on starch, maltose, peptone and complex organic substrates. Only CO₂ and H₂ could be detected as metabolic products, the latter being inhibitory to growth at high concentrations. Hydrogen inhibition can be prevented by the addition of S^o, whereupon H₂S is formed in addition, most likely as the result of a detoxification reaction. The GC-content of the DNA of isolate Vc 1 is 38 mol%. The new genus is named Pyrococcus, the fireball. Type species and strain is Pyrococcus furiosus Vc 1 (DSM 3638).     

Failure of an iterative curve-fitting procedure to successfully estimate two organic N pools

Fitting a double negative exponential function to N mineralization data can be used to characterize two organic nitrogen pools; an easily decomposable (N_dpm) and a resistant one (N_rpm). The relevance of those two calculated N mineralization pools was investigated by adding easily decomposable organic material to soils. Soil amended with crop residues of sugar-beet or bean was mixed with an equal amount of coarse sand, incubated at 35 °C and leached at specific time-intervals. Upon leaching, NH₄ ⁺ and NO₃ ^- were measured in the extracts. A double negative exponential function was fitted to the data and two organic N pools were defined. Fitting a double negative exponential function to N mineralization data to characterize an active and resistant organic N pool was sometimes impossible; the N mineralization data did not always resemble a negative exponential function. Additionally, the size of the two pools calculated were not constant with time and were often meaningless; the N_rpm pool was greater than the soil organic N content, the size of the N_rpm pool was smaller than the N_dpm pool or one of the N pools was negative. Relevant values for both N_rpm and N_dpm which were consistent with incubation time were only obtained when excessive amounts of organic material, normally not dealt with in the field, were applied.