Effects of low temperature and freeze-thaw cycles on hydrocarbon biodegradation in Arctic tundra soil.

Degradation of petroleum hydrocarbons was monitored in microcosms with diesel fuel-contaminated Arctic tundra soil incubated for 48 days at low temperatures (-5, 0, and 7 degrees C). An additional treatment was incubation for alternating 24-h periods at 7 and -5 degrees C. Hydrocarbons were biodegraded at or above 0 degrees C, and freeze-thaw cycles may have actually stimulated hydrocarbon biodegradation. Total petroleum hydrocarbon (TPH) removal over 48 days in the 7, 0, and 7 and -5 degrees C treatments, respectively, was 450, 300, and 600 microg/g of soil. No TPH removal was observed at -5 degrees C. Total carbon dioxide production suggested that TPH removal was due to biological mineralization. Bacterial metabolic activity, indicated by RNA/DNA ratios, was higher in the middle of the experiment (day 21) than at the start, in agreement with measured hydrocarbon removal and carbon dioxide production activities. The total numbers of culturable heterotrophs and of hydrocarbon degraders did not change significantly over the 48 days of incubation in any of the treatments. At the end of the experiment, bacterial community structure, evaluated by ribosomal intergenic spacer length analysis, was very similar in all of the treatments but the alternating 7 and -5 degrees C treatment.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

Cyclic variations in nitrogen uptake rate in soybean plants

Uptake of NO₃⁻ by nonnodulated soybean plants (Glycine max L. Merr. cv Ransom) growing in flowing hydroponic culture at 22 and 14°C root temperatures was measured daily during a 31-day growth period. Ion chromatography was used to determine removal of NO₃⁻ from solution during each 24-hour period. At both root-zone temperatures, rate of NO₃⁻ uptake per plant oscillated with a periodicity of 3 to 5 days. The rate of NO₃⁻ uptake per plant was consistently lower at 14°C than 22°C. The lower rate of NO₃⁻ uptake at 14°C during the initial 5 to 10 days was caused by reduced uptake rates per gram root dry weight, but with time uptake rates per gram root became equal at 14 and 22°C. Thereafter, the continued reduction in rate of NO₃⁻ uptake per plant at 14°C was attributable to slower root growth.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

Combinations of Intervention Treatments Resulting in 5-Log10-Unit Reductions in Numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 Organisms in Apple Cider

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log₁₀ units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log₁₀ units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log₁₀-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log₁₀-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log₁₀-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil

A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (10³ to 10⁴ CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced Acinetobacter baumannii strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.     

Rapid increase in deep supercooling of xylem parenchyma

Malus pumila Mill. twigs were collected from September through December and stored at 5°C until the low temperature exotherms of the xylem were determined by differential thermal analysis. During the differential thermal analysis, cooling was interrupted, and temperatures of 5 to −18°C were held for 0.4 to 10 hours before cooling to −50°C was resumed. Control twigs were cooled to −50°C without interruption. Holding the twigs at 1.3 to −5°C shifted the start of the low temperature exotherm from about −20 to −30°C. Slightly higher (2.6°C) and lower (−10°C) temperatures were occasionally effective. The shift began within 20 to 30 minutes and increased progressively to 150 minutes. The acclimation was reversibly inhibited by N₂ atmosphere.     

Ice Nucleation Activity in Lichens

A newly discovered form of biological ice nucleus associated with lichens is described. Ice nucleation spectra of a variety of lichens from the southwestern United States were measured by the drop-freezing method. Several epilithic lichen samples of the genera Rhizoplaca, Xanthoparmelia, and Xanthoria had nuclei active at temperatures as warm as −2.3°C and had densities of 2.3 × 10⁶ to more than 1 × 10⁸ nuclei g⁻¹ at −5°C (2 to 4 orders of magnitude higher than any plants infected with ice nucleation-active bacteria). Most lichens tested had nucleation activity above −8°C. Lichen substrates (rocks, plants, and soil) showed negligible activity above −8°C. Ice nucleation-active bacteria were not isolated from the lichens, and activity was not destroyed by heat (70°C) or sonication, indicating that lichen-associated ice nuclei are nonbacterial in origin and differ chemically from previously described biological ice nuclei. An axenic culture of the lichen fungus Rhizoplaca chrysoleuca showed detectable ice nucleation activity at −1.9°C and an ice nucleation density of 4.5 × 10⁶ nuclei g⁻¹ at −5°C. It is hypothesized that these lichens, which are both frost tolerant and dependent on atmospheric moisture, derive benefit in the form of increased moisture deposition as a result of ice nucleation.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

The Control of Growth Habit of Marquillo x Kenya Farmer Wheat Dwarf I by Temperature

The Marquillo × Kenya Farmer 1 “grass-clump” dwarf selection of Triticum aestivum L. was grown under continuous 2000 foot candle light and several regimes of alternating 16° and 26° temperatures combined in total cycle lengths of 6, 12, 24, or 48 hr. Plants at 26° grew as normal wheat. Those exposed to 0.25 to 2 hr of 16° per cycle showed typical “grass-clump” dwarf characteristics which were independent of the cycle length. Treatments with 16° exposures of 4 to 8 hr per 24 hr and 12 to 16 hr per 48 hr exhibited vegetative “grass-clump” dwarfness for 40 days but later displayed extensive reproductive development. Longer 16° treatments killed the plants at a very early stage of vegetative development before floral initiation. The data supported an hypothesis that all 4 growth habits were related to the temperature sensitivity of the vegetative meristem. The cessation of meristem development was possibly due to the accumulation of a stable inhibitory substance produced at low temperatures.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Ethylene Removal at Low Temperatures under Biofilter and Batch Conditions

Removal of the plant hormone ethylene (C₂H₄) is often required by horticultural storage facilities, which are operated at temperatures below 10°C. The aim of this study was to demonstrate an efficient, biological C₂H₄ removal under such low-temperature conditions. Peat-soil, acclimated to degradation of C₂H₄, was packed in a biofilter (687 cm³) and subjected to an airflow (~73 ml min⁻¹) with 2 ppm (μl liter⁻¹) C₂H₄. The C₂H₄ removal efficiencies achieved at 20, 10, and 5°C, respectively, were 99.0, 98.8, and 98.4%. This corresponded to C₂H₄ levels of 0.022 to 0.032 ppm in the biofilter outlet air. At 2°C, the average C₂H₄ removal efficiency dropped to 83%. The detailed temperature response of C₂H₄ removal was tested under batch conditions by incubation of 1-g soil samples in a temperature gradient ranging from 0 to 29°C with increments of 1°C. The C₂H₄ removal rate was highest at 26°C (0.85 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹), but remained at levels of 0.14 to 0.28 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹ at 0 to 10°C. At 35 to 40°C, the C₂H₄ removal rate was negligible (0.02 to 0.06 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹). The Q₁₀ (i.e., the ratio of rates 10°C apart) for C₂H₄ removal was 1.9 for the interval 0 to 10°C. In conclusion, the present results demonstrated microbial C₂H₄ removal, which proceeded at 0 to 2°C and produced a moderately psychrophilic temperature response.     

Metabolic Activity of Permafrost Bacteria below the Freezing Point

Metabolic activity was measured in the laboratory at temperatures between 5 and −20°C on the basis of incorporation of ¹⁴C-labeled acetate into lipids by samples of a natural population of bacteria from Siberian permafrost (permanently frozen soil). Incorporation followed a sigmoidal pattern similar to growth curves. At all temperatures, the log phase was followed, within 200 to 350 days, by a stationary phase, which was monitored until the 550th day of activity. The minimum doubling times ranged from 1 day (5°C) to 20 days (−10°C) to ca. 160 days (−20°C). The curves reached the stationary phase at different levels, depending on the incubation temperature. We suggest that the stationary phase, which is generally considered to be reached when the availability of nutrients becomes limiting, was brought on under our conditions by the formation of diffusion barriers in the thin layers of unfrozen water known to be present in permafrost soils, the thickness of which depends on temperature.     

Thermal denaturation in acidic solutions of double-helical ribonucleic acid from virus-like particles found in Penicillium chrysogenum. A spectrophotometric study

1. Two species of double-helical RNA isolated from mycelium of Penicillium chrysogenum were titrated with acid at 25°C and 95°C (solvent 0.1m-sodium phosphate buffer). At 25°C denaturation occurred at about pH3. At 95°C in the denatured form cytosine residues titrated as a simple monobasic acid of pK3.9 compared with pK2.5 for the native form at 25°C. 2. On thermal denaturation in neutral and acidic solutions one species of RNA (38% rG·rC) `melted' in three distinct stages, equivalent to a mixture of three species, namely one of about 25% rG·rC, another of about 33% rG·rC and a third of about 46% rG·rC: the relative proportions were 0.25:0.35:0.40. 3. On thermal denaturation in acidic solutions the increase in the fraction of ionized cytosine residues concomitant with the `melting' of rG·rC base pair also affects the spectrum especially at 280nm and serves to enhance the contribution of rG·rC base pairs at this wavelength. The increment in ε_(P) at 280nm on `melting' an rG·rC base pair approaches 53501·mol<sup>−1</sup>·cm<sup>−1</sup> depending on pH, compared with 33501·mol<sup>−1</sup>·cm<sup>−1</sup> at pH7. In contrast ε<sub>(P)</sub> at 280nm is scarcely affected by `melting' rA·rU base pairs or by the protonization of adenine residues. 4. Changes in the spectrum of Escherichia coli rRNA on denaturation in acidic solutions were studied to yield the mole fractions of rA·rU and rG·rC base pairs `melting' at particular pH values.     

Effect of cold hardening on sensitivity of winter and spring wheat leaves to short-term photoinhibition and recovery of photosynthesis

Photoinhibition of photosynthesis and its recovery were studied in wheat (Triticum aestivum L.) leaves grown at nonhardening (20°C) and cold-hardening (5°C) temperatures. Cold-hardened wheat leaves were less susceptible to photoinhibition at 5°C than nonhardened leaves, and the winter cultivars, Kharkov and Monopol, were less susceptible than the spring cultivar, Glenlea. The presence of chloramphenicol, a chloroplastic protein synthesis inhibitor, increased the susceptibility to photoinhibition, but cold-hardened leaves still remained less susceptible to photoinhibition than nonhardened leaves. Recovery at 50 μmol m⁻² s⁻¹ photosynthetic photon flux density and 20°C was at least biphasic, with a fast and a slow phase in all cultivars. Cold-hardened leaves recovered maximum fluorescence and maximum variable fluorescence in the dark-adapted state during the fast phase at a rate of 42% h⁻¹ compared with 22% h⁻¹ for nonhardened leaves. The slow phase occurred at similar rates (2% h⁻¹) in cold-hardened and nonhardened leaves. Full recovery required up to 30 h. Fast-recovery phase was not reduced by either lowering the recovery temperature to 5°C or by the presence of chloramphenicol. Slow-recovery phase was inhibited by both treatments. Hence, the fast phase of recovery does not require de novo chloroplast protein synthesis. In addition, only approximately 60% of the photochemical efficiency lost through photoinhibition at 5°C was associated with lost [¹⁴C]atrazine binding and, hence, with damage to the secondary quinone electron acceptor for photosystem II-binding site. We conclude that the decrease in susceptibility to photoinhibition exhibited following cold hardening of winter and spring cultivars is not due to an increased capacity for repair of photoinhibitory damage at 5°C but reflects intrinsic properties of the cold-hardened photosynthetic apparatus. A model to account for the fast component of recovery is discussed.     

Effect of sowing dates and vernalization on Betavulgaris L. cv. Univers C-leaf structure

This research was conducted to study the effect of three different sowing dates (15th October, 15th November and 15th December) and two vernalization treatments (5 °C and −20 °C) on leaf structure of Betavulgaris L. cv. Univers. The obtained data are summarized as follows:The maximum values of the most studied parameters; lower epidermis + spongy tissue thickness, midrib, mesophyll tissue, vascular bundle, collenchymatous tissue and number of xylem vessels per arm were found as a result of 15th October sowing date treatment compared with the two other sowing dates. Furthermore, effect of the cooling treatments varied according to the recorded character, sowing date and cooling degree. Most of the vernalization treatments at early sowing dates increased the mesophyll tissue, midrib, number of vascular bundles per transverse section, vascular bundle thickness and number of xylem arms per transverse section.The two studied cooling treatments at 15th October sowing date increased both stomatal index and average number of stomata: average number of epidermis cells compared with the control. Furthermore, 15th October under −20 °C treatment led to small epidermal cells and stomata formation, straight epidermal cell walls and closed stomata in comparison to the control.     

Survival of Plant Tissue at Super-Low Temperature VI. Effects of Cooling and Rewarming Rates on Survival

The survival rates of the cortical parenchymal cells of mulberry tree were determined as a function of cooling and rewarming rates. When cooling was carried out slowly at 1° to 15° per minute, all of the cells still remained viable even when rewarmed either rapidly or slowly. Survival rates gradually decreased to zero as the cooling rate increased from about 15° to 2000° per minute. In the intermediate cooling rates, when the cells were cooled at the rates lower than 14° per minute, from −2.2° to about −10°, these cells could survive subsequent rapid cooling and rewarming.

However, at cooling rates above 1000° per minute and with rapid rewarming, the effect of cooling rate reversed and survival increased, reaching a maximum at about 200,000° per minute. As the cooling rate increased above 15° per minute, survival rates became increasingly dependent on the rewarming rate, with rapid rewarming becoming less deleterious than slow rewarming.

The temperature range at which damage occurred during rewarming following removal from liquid nitrogen and in which growth rate of ice crystallization was greatest, was −30° to −40°. The survival rates even in the prefrozen cells at −30° decreased considerably by keeping them at −30° for 10 minutes after removal from liquid nitrogen. This fact indicates that intracellular freezable water remains to some degree even in the prefrozen cells at −30°. After removal from liquid nitrogen, all cells retained their viability, when they were passed rapidly through a temperature range between −50° and −2.5° within about 2 seconds, namely at the rates greater than 1000° per minute.

These observations are explained in terms of the size of the crystals formed within the cortical cells.

     

Prototheca zopfii Krüger Strain UMK-13 Growth on Acetate or n-Alkanes

A new strain of Prototheca zopfii Krüger was grown on acetate or on pure n-alkanes. A maximum acetate-supported exponential growth of 12 divisions day⁻¹ occurred at pH 5 and 30°C. At 25°C, growth on n-alkanes was almost as fast, but no growth occurred at 30°C. After 4 days at 25°C, 34 to 45% of the n-alkanes had been removed, whereas at 21°C and slower growth, utilization was twofold greater after 15 days. Rates of growth and utilization increased markedly after a point of sudden emulsification.     

Enzymes in cancer: Asparaginase from chicken liver

1. A procedure for partial purification of asparaginase from chicken liver is presented. 2. The bulk of the enzyme is located in the soluble fraction of chicken liver. 3. Molecular weights of chicken-liver asparaginase and of the guinea-pig serum enzyme, estimated by gel filtration, were 306000 and 210000 respectively. The Michaelis constants (K_m) at 37° and pH8·5 were 6·0×10⁻⁵m and 7·2×10⁻⁵m respectively. 4. At 50° the chicken-liver enzyme was moderately stable, some activity being lost by aggregation; in dilute electrolyte solutions the activity rapidly diminished. 5. The anti-lymphoma effect of guinea-pig serum in mice carrying the 6C3HED tumour was confirmed. Chicken-liver asparaginase also showed an effect but in this case the enzyme preparation had to be administered repeatedly. 6. Guinea-pig serum asparaginase was stable for several days in mouse blood, after intraperitoneal injection, whereas chicken-liver asparaginase rapidly disappeared. 7. Aspartic acid β-hydrazide was shown to be a competitive inhibitor of chicken-liver asparaginase with K_i approx. 5·6×10⁻⁴m. In mice it produced an anti-lymphoma effect, as reported previously.