In situ mineralization of nitorgen and phosphorus of arctic soils after perturbations simulating climate change

Seasonal net nitrogen (N) and phosphorus (P) mineralization was investigated at Abisko, Swedish Lapland in soils of a subarctic heath and in soils of a colder (by about 4° C), high altitude fellfield by (a) using in situ soil incubation in soils which had been shaded or subjected to two levels of increased temperature, combined with (b) reciprocal transplantation of soils between the two sites. Proportionally large and significant net seasonal mineralization of N, in contrast to non-significant P mineralization, was found in untransplanted and transplanted fellfield soil. In contrast, P was mineralized in proportionally large amounts, in contrast to low N mineralization, in the transplanted and untransplanted heath soil. The differences indicate that P was strongly immobilized in relation to N at the fellfield and that N was more strongly immobilized than P in the heath soil. The immobilization in both soils remained high even after a temperature change of 4–5° C experienced by transplanted soils. Air temperature increases of up to 4–5° C in greenhouses resulted in a soil temperature increase of 1–2° C and did not cause any extra increase of net N and P mineralization. The results suggest that soil temperature increases of up to 2° C, which are likely to occur by the end of the next century as an effect of a predicted 4–5° C rise in air temperature, have only small effects on net mineralization in at least two characteristic tundra soils. These effects are probably smaller than the natural fluctuation of plant available nutrients from site to site, even within the same plant community. A further soil temperature increase of up to 4–5° C may enhance decomposition and gross mineralization, but the rate of net mineralization, and hence the change of nutrient availability to the plants, depends on the extent of microbial immobilization of the extra nutrients released.     

Kinetic parameters of nitrogen mineralization rates of leguminous crops incorporated into soil

Summary Fresh leguminous plant residues were incorporated into soil columns and incubated at 23°C for up to 20 weeks. The N released from specific fractions (foliage, stems, and roots) of each residue were monitored at specific time intervals. Relationships between organic carbon, total nitrogen, CN ratio, lipids, and lignin content of the plant materials and the cumulative amount of N mineralized in soil were investigated. Statistical analyses indicated that the rates of N mineralized were not significantly correlated with the organic C nor lipid content of the residues. However, the cumulative amount of N released was significantly correlated with the total N content of the plant material (r=0.93^***). The percentage of organic N of the legumes mineralized in soil ranged from 15.9 to 76.0%. The relationship between the percentage of N released and the CN ratio of the plant material showed an inverse cuvilinear response (r= 0.88^***). It was also evident that the composition of lignin in the residue influenced N mine-ralization rates of the leguminous organs incorporated into soil.There was a curvilinear relationship between the cumulative amount of N released from the residues and time of incubation. Nitrogen mineralization rates were described by first-order kinetics to estimate the N mineralization potential (N₀), mineralization rate constant (k), and the time of incubation required to mineralize one-half of N₀ (t_1/2). The kinetic parameters were calculated by both the linear least squares (LLS) and nonlinear least squares (NLLS) transformations. The N₀ values among the crop residues varied from –35 to 510 g Ng^–1 soil. Statistical analyses revealed that the N₀ values obtained by both LLS and NLLS methods were significantly correlated (r=0.93^***). The mineralization rate constants (k) of the residues ranged from 0.045 to 0.325 week^–1. The time of incubation required to mineralize one-half the nitrogen mineralization potential (t_1/2) of the legumes incorporated into soil ranged from 2.1 to 15.4 weeks.     

Mineralization of labeled N from cowpea [Vigna unguiculata (L.) Walp.] plant parts at two growth stages in sandy soil

Cowpea [Vigna unguiculata (L). Walp.] has great potential as green manure due to its rapid N accumulation and efficient N₂ fixation. The objective of this study was to measure the rate of N mineralization from cowpea plant parts harvested at onset of flowering (5 weeks) and mid pod-fill (7 weeks) under near optimum conditions. Cowpeas were grown in a greenhouse and supplied with ¹⁵NH₄ ¹⁵NO₃ to isotopically label tissue. Cowpea leaves, stems, and roots were incorporated into a sandy soil (Psammentic Paleustalf) and net N mineralized was measured several times during a 10 week incubation. The amount of N accumulated in 7-week old cowpeas was more than double that in 5-week old cowpeas. The portion of N mineralized after 10 weeks was 24% for 5-week old cowpeas and 27% for 7-week old cowpeas. The rate of N mineralization from leaves and stems increased with plant age, but decreased for roots. The amount of N mineralized from 7-week old cowpeas was more than double (235%) that from 5-week old cowpeas due to greater N accumulation and a more rapid rate of N mineralization of the more mature cowpeas. The greatest amount of N was released from leaves, which amounted to 74 and 65% of total N mineralization from 5- and 7-week old cowpeas, respectively. The percentage of N mineralized by 10 weeks was linearly related to the tissue N concentration of the plant parts and to their C/N ratio. These relationships allow a quick estimation of the amount of N that would mineralize from cowpea residues incorporated into soil based on their N concentration or C/N ratio.     

Net mineralization and nitrification rates in a clay soil measured and predicted in permanent grassland from soil temperature and moisture content

Summary Net mineralization of N and net nitrification in field-moist clay soils (Evesham-Kingston series) from arable and grassland sites were measured in laboratory incubation experiments at 4, 10 and 20°C. Three depth fractions to 30 cm were used. Nitrate accumulated at all temperatures except when the soil was very dry (=0.13 cm³ cm^–3). Exchangeable NH₄-ions declined during the first 24 h and thereafter remained low. Net mineralization and net nitrification approximated to zero-order reactions after 24 h, with Q₁₀ values generally <1.6. The effect of temperature on both processes was linear although some results conformed to an Arrhenius-type relationship. The dependence of net mineralization and net nitrification in the field soil on soil temperature (10 cm depth) and moisture (0–15, 15–25, 25–35 cm depths) was modelled using the laboratory incubation data. An annual net mineralization of 350 kg N ha^–1 and net nitrification of 346 kg N ha^–1 were predicted between September 1980 and August 1981. The model probably overstressed the effect of soil moisture relative to soil temperature.     

Soil nitrogen mineralization in plantations ofJuglans nigra interplanted with actinorhizalElaeagnus umbellata orAlnus glutinosa

Nitrogen mineralization rates were estimated in 19-year-old interplantings of black walnut (Juglans nigra L.) with dinitrogen fixing autumn-olive (Elaeagnus umbellata Thunb.) or black alder (Alnus glutinosa L. Gaertn.) and in pure walnut plantings at two locations in Illinois USA. N mineralization rates were measured repeatedly over a one year period usingin situ incubations of soil cores in oxygen-permeable polyethylene bags at 0–10 and 10–20 cm soil depths, and also by burying mixed-bed ion-exchange resin in soil. Mineralization rates were highest in summer and in plots containing actinorhizal Elaeagnus and Alnus in contrast with pure walnut plots. Elaeagnus plots at one location yielded 236 kg of mineral N ha^–1 yr^–1 in the upper 20 cm of soil, a value higher than previously reported for temperate decidous forest soils in North America. The highest mean plot values for N mineralization in soil at a location were 185 kg ha^–1 yr^–1 for Alnus interplantings and 90 kg ha^–1 yr^–1 for pure walnut plots. Plots which had high N mineralization rates also had the largest walnut trees. Despite low pH (4.1 and 6.5) and low extractable P concentrations (1.4 and 0.7 mg kg^–1 dry mass) at the two locations, nitrification occurred in all plots throughout the growing season. NO ₃ ^– –N was the major form of mineralized N in soil in the actinorhizal interplantings, with NH ₄ ⁺ –N being the major form of mineral N in control plots. Walnut size was highly correlated with soil nitrogen mineralization, particularly soil NO ₃ ^– –N production in a plot.     

13C isotope discrimination correlates with biological nitrogen fixation in soybean (Glycine max (L.) Merrill)

Ammonium sulphate was applied at the rate of 300 kg N ha^–1 with or without the nitrification inhibitor 1-carbamoyl-3(5)-methylpyrazol (4 kg ha^–1) to plots measuring 1.5 × 1.5 m. The fertilizer and the inhibitor were washed into the top 15-cm layer of the soil, which was highly calcareous (55% CaCO₃), and the plots were kept bare. The process of nitrification was monitored by regular soil sampling. In the absence of the inhibitor, nitrification was completed in three weeks. In the presence of the inhibitor only 10% of applied N was nitrified by the end of the third week and 42% by the end of the eighth week. Average soil temperature at 5–, 10– and 20-cm depth over the first six weeks was 26.0, 24.8 and 24.2°C, respectively.     

A comparison of organic and inorganic nitrogen fertilizers: Their nitrate-N and ammonium-N release characteristics and effects on the growth response of lettuce (Lactuca sativa L. cv. Fortune)

The response of pot grown lettuce to inorganic (ammonium nitrate) and organic (dried blood and Protox) N fertilizers was determined at two temperature regimes (15°C day/10°C night and 20°C day/15°C night) and related to the NH₄–N and NO₃–N release characteristics of each material. The N release characteristics of the organic materials matched the N requirements of lettuce more closely than the inorganic fertilizer. However, was rapidly released from the protein based materials such that composts were depleted of available fertilizer N at the same time irrespective of the form supplied. The warmer temperature regimes resulted in a more rapid depletion of the fertilizers due to biological immobilization such that N recoveries in shoots, roots and leachates were reduced. Approximately 20% of the N present in Protox (a material derived from activated sewage sludge, processed to reduce the heavy metal content to minimal levels) appeared to be resistant to microbial degradation and was unavailable to the plants. Therefore, the growth response of lettuce was slightly reduced with Protox compared to the other materials at similar rates of incorporation. The organic materials did not contribute NO₃–N to the plant and small NO₃–N concentrations in petioles were derived from the water used for irrigation. However, NO₃–N levels in plants receiving inorganic ammonium nitrate were initially high but progressively declined as the fertilizer NO₃–N became depleted.     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060–5010 mg/kg dry wt of culture) at ambient temperature (16–29°C) in daylight than at ambient temperature (18–23 °C) in darkness or at controlled temperatures of 11 °C, 20 °C or 25 °C in darkness. Yields at 25 °C were low. Incubation at 11 °C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 °C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 °C (2460-21 360 mg/kg), 1 strain at 11 °C (6570 mg/kg). Yields at a temperature oscillating daily from 10–20 °C were less than at 15 °C. One of the 5 strains produced appreciable amounts of a-zearalenol (1645 mg/kg at 20°C) and 2 of nivalenol (340 and 499 mg/kg at 20 °C).     

Effect of temperature on growth and nitrogen mineralization of fungi and fungal-feeding nematodes

Dish and column microcosms containing alfalfa-sand medium were established to determine effect of temperature on growth and N mineralization ability of two fungi (Rhizoctonia solaniand Botrytis cinerea) and two nematodes (Aphelenchus avenaeand Aphelenchoides composticola). The microcosms were incubated at 15, 20, 25 and 29 °C for 21 days. In the dish microcosms, hyphal growth rates of both fungal species increased with temperature in the range of 15–25 °C. Above that temperature range, the growth rate of R. solani remained almost constant while that of B. cinereadecrease considerably. The population growth rate of A. avenae increased with temperature between 15 and 29 °C on colonies of R. solani and B. cinerea in dish microcosms. The growth rate of A. composticola also increased in the range of 15–25 °C but decreased greatly beyond that temperature range independent of the fungal species as food source. Inorganic N (NH₄ ⁺ + NO₃ ^–) was collected from each column microcosm by leaching every 3 days. In the columns containing R. solani, there was a significant effect of temperature on the amount of N detected in the fungus+A avenaeor A. composticolabut not in the fungus alone columns. The total amount of N was greatest at 29 °C for A. avenaeand at 20 °C for A. composticola columns, concurrent with the population growth rates of the nematodes. In the columns containing B. cinerea, the effect of temperature on the amount of inorganic N was not significant in either the fungus alone or fungus+nematode columns, although the population growth rates of the both nematode species were highest at 20 °C. For B. cinerea, the N amount across temperatures was the same or larger for the fungus alone as for the fungus+nematode columns. In general, the contribution of fungal-feeding nematodes to N mineralization was small in any combinations of fungus and nematode species at any temperature. Similarity in C/N ratio of the fungal and nematode biomass, organic substrate C/N ratios too low for measurable increase in net mineralization by the nematodes and small reproduction of the nematodes in the column microcosms were probable contributory factors.     

The kinetics of the degradation of chloroform and benzene in anaerobic sediment from the River Rhine

In anaerobic methanogenic sediment microcosms¹⁴C labelled chloroform was degraded mainly to carbon dioxide. At a concentration of 4 g.l^–1 the mineralization followed first order kinetics with a half life of 12 days at 10°C and 2.6 days at 20°C. At a concentration of 400 g.l^–1 the mineralization rate increased with time and followed logarithmic kinetics with a _max of 0.02.d^–1 at 10°C. The logarithmic kinetics can be explained by growth of the bacteria on the higher concentration of chloroform with a generation time of 35 days. Shaking and oxygenation did not inhibit the mineralization of chloroform, probably because of bacterial consumption of the dissolved oxygen. ¹⁴C labelled benzene was mineralized only for a small percentage to¹⁴C labelled carbon dioxide while other, not acid extractable, degradation products were formed. Under anaerobic conditions after one day when 5% of the benzene was degraded to carbon dioxide, the mineralization ceased, while the disappearance of benzene proceeded. With air in the headspace of the incubation bottles 25% of the benzene was mineralized to carbon dioxide. The anaerobic degradation of benzene at a concentration of 100 .l^–1 showed similar kinetics as the degradation at 1 g.l^–1. Hence no adaptation of the microflora in the sediment occurred during the 63 days of the experiment at 100 g.l^–1.     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

Effect of temperature on humus respiration rate and nitrogen mineralization: Implications for global climate change

Respiration and nitrogen mineralization rates of humus samples from 7 Scots pine stands located along a climatic transect across the European continent from the Pyrenees (42°40) to northern Sweden (66°08) were measured for 14 weeks under laboratory conditions at temperatures from 5 °C to 25 °C. The average Q₁₀ values for the respiration rate ranged from about 1.0 at the highest temperature to more than 5 at 10 °C to 15 °C in the northernmost samples. In samples from more northern sites, respiration rates remained approximately constant during the whole incubation period; in the southern end of the transect, rates decreased over time. Respiration rate was positively correlated with incubation temperature, soil pH and CN ratio, and negatively with soil total N. Regressions using all these variables explained approximately 71% of the total variability in the respiration rate. There was no clear relation between the nitrogen mineralization rate and incubation temperature. Below 15 °C the N-mineralization rate did not respond to increasing temperature; at higher temperatures, significant increases were found for samples from some sites. A regression model including incubation temperature, pH, N_tot and CN explained 73% of the total variability in N mineralization. The estimated increase in annual soil respiration rates due to predicted global warming at the high latitudes of the Northern Hemisphere ranged from approximately 0.07×10¹⁵ to 0.13×10¹⁵ g CO₂ at 2 °C and 4 °C temperature increase scenarios, respectively. Both values are greater than the current annual net carbon storage in northern forests, suggesting a switch of these ecosystems from net sinks to net sources of carbon with global warming.     

Soil and fertilizer nitrogen transformations under alternate flooding and drying moisture regimes

Summary A study of changes in NH₄ ⁺ and NO₃ ^––N in Maahas clay amended with (NH₄)₂SO₄ and subjected to 4 water regimes in the presence and absence of the nitrification inhibitor N-Serve (Nitrapyrin) showed that the mineral N was well conserved in the continoous regimes of 50% and 200% (soil weight basis) but suffered heavy losses due to nitrification-denitrification under alternate drying and flooding. N-Serve was effective in minimizing these losses.Another incubation study with 3 soils showed that after 10 cycles of flooding and drying (either at 60°C or 25°C), the ammonification of soil N was enhanced. Nitrification of soil as well as fertilizer NH₄ ⁺ was completely inhibited upto 4 weeks by the treatments involving drying at high temperature. Flooding and air drying at 25°C, on the other hand, enhanced ammonification of soil N but retarded nitrification. These treatments, however, enhanced both ammonification and nitrification of the applied NH₄ ⁺ fertilizer N. Under flooded conditions rate of NH₄ ⁺ production was faster in soils that were dried at 60°C or 25°C and then flooded as compared to air dried soils.It is concluded that N losses by nitrification-denitrification and related N transformations may be considerably altered by alternating moisture regimes. Flooding and drying treatments seem to retard nitrification of soil N but conserve that of fertilizer NH₄ ⁺ applied after these treatments.     

Gellan gum gel as entrapment matrix for high temperature fermentation processes: A rheological study

High temperature rheological measurements (60–80°C) were carried out on gellan gum gel. The experiments were conducted in order to optimize the behavior of the gel as an entrapment matrix for fermentation processes of dairy fluids using highly thermophilic microorganisms. At 60°C, no significant decrease of rheological parameters of the gel was observed for 15 days, as compared to 23°C. At 80°C, the gel weakened abruptly. The gel was extremely sensitive to calcium loss (optimum concentration 0.1% CaCl₂) due to chelating agents. Gellan gum gel could be an efficient entrapment matrix for fermentation processes at temperature up to 60°C in dairy fluids.     

Nitrogen mineralization dynamics in grass monocultures

Although Wedin and Tilman (1990) observed large differences in in situ N mineralization among monocultures of five grass species, the mechanisms responsible were unclear. In this study, we found that the species did not change total soil C or N, and soil C: N ratio (range 12.9–14.1) was only slightly, but significantly, changed after four years. Nor did the species significantly affect the total amount of N mineralized (per g soil N) in year-long aerobic laboratory incubations. However, short-term N mineralization rates in the incubations (day 1–day 17) differed significantly among species and were significantly correlated with annual in situ mineralization. When pool sizes and turnover rates of potentially mineralizable N (N_o) were estimated, the best model treated N_o as two pools: a labile pool, which differed among species in size (N_l, range 2–3% of total N) and rate constant (h, range 0.04–0.26 wk^–1), and a larger recalcitrant pool with a constant mineralization rate across species. The rate constant of the labile pool (h) was highly correlated with annual in situ N mineralization (+0.96). Therefore, plant species need only change the dynamics of a small fraction of soil organic matter, in this case estimated to be less than 3%, to have large effects on overall system N dynamics.     

Relationships between soil microbial properties and aboveground stand characteristics of conifer forests in Oregon

Eight forest sites representing a large range of climate, vegetation, and productivity were sampled in a transect across Oregon to study the relationships between aboveground stand characteristics and soil microbial properties. These sites had a range in leaf area index of 0.6 to 16 m² m^–2 and net primary productivity of 0.3 to 14 Mg ha^–1 yr^–1.Measurements of soil and forest floor inorganic N concentrations and in situ net N mineralization, nitrification, denitrification, and soil respiration were made monthly for one year. Microbial biomass C and anaerobic N mineralization, an index of N availability, were also measured. Annual mean concentrations of NH ₄ ⁺ ranged from 37 to 96 mg N kg^–1 in the forest floor and from 1.7 to 10.7 mg N kg^–1 in the mineral soil. Concentrations of NO ₃ ^– were low ( < 1 mg N kg^–1) at all sites. Net N mineralization and nitrification, as measured by the buried bag technique, were low on most sites and denitrification was not detected at any site. Available N varied from 17 to 101 mg N kg^–1, microbial biomass C ranged from 190 to 1230 mg Ckg^–1, and soil respiration rates varied from 1.3 to 49 mg C kg^–1 day^–1 across these sites. Seasonal peaks in NH ₄ ⁺ concentrations and soil respiration rates were usually observed in the spring and fall.The soils data were positively correlated with several aboveground variables, including leaf area index and net primary productivity, and the near infrared-to-red reflectance ratio obtained from the airborne simulator of the Thematic Mapper satellite. The data suggest that close relationships between aboveground productivity and soil microbial processes exist in forests approaching semi-equilibrium conditions.Abbreviations IR infrared - LAI leaf area index - k _c proportion of microbial biomass C mineralized to CO₂ - NPP net primary productivity - TM Thematic Mapper     

Factors affecting broth viscosity and coenzyme Q10 production by Agrobacterium species

Summary In the production of coenzyme Q₁₀ (CoQ₁₀) by Agrobacterium sp. the culture broth becomes highly viscous. In an attempt to improve the production process, the effects of chemical and physical factors on broth viscosity and CoQ₁₀ production were studied, using Agrobacterium sp. KY-8593. A particular concentration ratio of sugar to ammonium-nitrogen (NH₄–N) in the medium could effectively enhance CoQ₁₀ production without increasing broth viscosity. An increase in culture temperature to between 32°C and 34°C lowered broth viscosity without reducing CoQ₁₀ production. NH₄–N concentration and temperature had a correlative effect on broth viscosity. At a temperature of about 33°C, there was a wide range of NH₄–N concentration which was optimal for both broth viscosity and CoQ₁₀ production. In optimal conditions with 8% sugar the apparent broth viscosity was reduced to less than 10 pseudo-cP and CoQ₁₀ production was increased to more than 80 mg/l.     

Patern of nitrogen release during decomposition of some green manures in a tropical alluvial soil

Summary The pattern of release of ammonium and nitrate nitrogen during decomposition of glyricidia, sunflower, centrosema, calapagonium and crotolaria under aerobic and anaerobic conditions, in an alluvial soil over a period of 7 weeks was studied. Under aerobic conditions, the NH₄ ⁺–N production reached the maximum after the 4th week. Nitrate-N and total available-N increased in all cases throughout the incubation period except in sunflower. This showed a nitrification inhibitory effect and had a relatively high C/N ratio (11.0) and low total N content (2.8%). In general the increase in NH₄ ⁺–N and NO₃ ^––N was more rapid in the early stages of incubation.Under anaerobic conditions, the production of these nutrients was considerably low. Soil organic matter mineralized faster than the added organic material which started to decompose slowly after sometime. Nitrate-N tend to decrease during incubation attributable to denitrification.     

EUF-analysis of tea soils of Southern India and tea productivity

Summary The relationship between EUF extractable nutrients and conventional soil test extractable nutrients in the acid soils of Southern India on one hand and that between EUF values and tea productivity on the other are described. Close correlation exists between EUF-NO₃–N at 20°C and CuSO₄–Ag₂SO₄-extractable NO₃–N (r=0.98^***), EUF-N_org and Morgan's reagent extractable NH₄–N (r=0.97^***), total EUF-N and CuSO₄–Ag₂SO₄-extractable NO₃–N plus Morgan's reagent NH₄–N (r=0.96^***), EUF-P at 20°C and modified Bray II-P (r=0.93^***) and EUF-P at 20°C plus that at 80°C and modified Bray II-P (r=0.91^***). The EUF-K at 20°C shows close correlation with NH₄OAc–K (r=0.80^***), Ag-thiourea-K (r=0.86^***) and Morgan's reagent-K (r=0.84^***) whereas the EUF-K at 80°C shows close correlation with the difference in K contents of NH₄OAc–K and Ag-thiourea-K (r=0.92^***) or of NH₄OAc–K and Morgan's reagent-K (r=0.93^***) and fixed NH₄–N (r=0.89^***). EUF-Ca, EUF-Mg and EUF-Mn do not show any relationship with conventional soil test values. Tea productivity is strongly associated with EUF-N and EUF-P extracted at 20°C.     

Behaviour of proteolytic Clostridium botulinum types A and B near the lower temperature limits of growth

Summary The behaviour of spores of Clostridium botulinum type A and proteolytic C. botulinum type B has been studied in cooked meat medium at 10°C, 12°C, 15°C, and 20°C, using mixed cultures (9 groups of in total 41 strains) and pure cultures (41 strains).At 10°C a decrease of 1–1.5 log cycles for type B and of 2–4 log cycles for type A Clostridia was observed. Neither growth nor toxin formation could be demonstrated.At 12°C spores of some strains developed and formed toxin with 3–4 weeks, whereas other strains did not develop within 7 weeks.At 15°C growth and toxin formation could be observed within 1 week, whereas at 20°C toxin was formed mostly within 2 or 3 days. Incubation at 10°C prior to incubation at 20°C seemed to have some effect on the lag time.