Use of high-gradient magnetic fishing for reducing proteolysis during fermentation

Proteolysis during fermentation may have a severe impact on the yield and quality of a secreted product. In the current study, we demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation. Bacitracin-linked magnetic affinity adsorbents were employed directly in a fermenter during Bacillus licheniformis cultivation to remove trace amounts of unwanted proteases. The constructed magnetic adsorbents had excellent, highly specific binding characteristics in the fermentation broth (K(d) = 1.94 micromolar; Q(max) = 222.8 mg/g), which obeyed the Langmuir isotherm and had rapid binding kinetics (equilibrium in <300 s). When applied directly in shake-flask cultures or in a 1-L fermenter and then removed by HGMF, the degradation of the model protein bovine serum albumin was stopped. The adsorbents could be recycled and reused during the same fermentation to remove freshly produced proteases, extending the life of the model protein in the fermenter. HGMF may provide an efficient method of stabilizing heterologous proteins produced in cultivation processes.     

Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine-activated killer cells reduces tumor load and prolongs survival in murine models

The control of malignancy disseminated within the peritoneal cavity is an important problem in the management of low-grade gastrointestinal and ovarian neoplasms. A model of peritoneal carcinomatosis in the mouse was used to investigate the potential of lymphokine-activated killer (LAK) cells and exogenous interleukin 2 (IL-2) to control intraperitoneal tumor. LAK cells are splenocytes activated in vitro by IL-2. C57BL/6 mice were injected intraperitoneally with a lethal inoculum of syngeneic MCA-105 tumor. Three days later, the established tumor was treated with adoptively transferred LAK cells and/or exogenous IL-2 administration. LAK cells alone were ineffective in reducing intraperitoneal tumor. Administration of IL-2 alone resulted in limited tumor reduction. Treatment with exogenous IL-2 in conjunction with LAK cells resulted in the greatest reduction of intraperitoneal tumor. The larger the number of LAK cells given, the greater the reduction in tumor. Frequent intraperitoneal bolus administration of IL-2 was more effective than a single daily intraperitoneal injection and intraperitoneal administration of IL-2 and LAK was more effective than systemic treatments. Marked prolongation of life was seen in mice treated with LAK cells plus exogenous IL-2. We conclude that intraperitoneal LAK cells plus exogenous IL-2 is an effective treatment regimen for reducing intraperitoneal tumor in this murine model.     

Nitrogen uptake and recovery from urea and green manure in lowland rice measured by15N and non-isotope techniques

In the recent past considerable attention is paid to minimize dependence on purchased inputs such as inorganic nitrogen fertilizer. Green manure in the form of flood-tolerant, stem-nodulatingSesbania rostrata andAeschynomene afraspera is an alternative N source for rice, which may also increase N use efficiency. Therefore research was conducted to determine the fate of N applied to lowland rice (Oryza sativa L.) in the form ofSesbania rostrata andAeschynomene afraspera green manure and urea in two field experiments using¹⁵N labeled materials.¹⁵N in the soil and rice plant was determined, and¹⁵N balances established. Apparent N recoveries were determined by non-tracer method. ¹⁵N recoveries averaged 90 and 65% of N applied for green manure and urea treatments, respectively. High partial pressures of NH₃ in the floodwater, and high pH probably resulted from urea application and favoured losses of N from the urea treatment. Results show that green manure N can supply a substantial proportion of the N requirements of lowland rice. Nitrogen released fromSesbania rostrata andAeschynomene afraspera green manure was in synchrony with the demand of the rice plant. The effect of combined application of green manure and urea on N losses from urea fertilizer were also investigated. Green manure reduced the N losses from¹⁵N labeled urea possibly due to a reduction in pH of the floodwater. Positive added N interactions (ANIs) were observed. At harvest, an average of 45 and 25% of N applied remained in the soil for green manure and urea, respectively.Contribution from IRRI, Los Baños, Philippines and Justus-Liebig-University, Giessen, GermanyContribution from IRRI, Los Baños, Philippines and Justus-Liebig-University, Giessen, Germany     

Iron reduction and gley formation by nitrogen-fixing clostridia

Summary Studies on iron reduction and the mechanism of gley formation by nitrogen-fixing clostridia are reported. Up to 10⁶ cells/g soil of anaerobic, nitrogen-fixing clostridia, capable of reducing iron (III)-oxide, were counted in samples taken from various top soils. In a gleyed subsoil as many as 10⁵ bacteria per g soil, capable of reducing and fixing nitrogen, were enumerated using the most probable number technique. In general, the ratio of the auxotrophic iron reducing clostridia (glucose+yeast extract fermenters) to the prototrophic iron reducing flora (glucose fermenters) was found much larger in the top soil samples than in those derived from various gleyed subsoils.An enrichment method for the isolation of nitrogen-fixing, iron reducing clostridia of the butyric-butyl type is described. The iron reducing capacity of this type of clostridia as well as of Clostridium pasteurianum was determined quantitatively. Generally, the presence of soil or soil extract enhanced the amount of dissolved ferrous iron, both with butyric acid fermenters and with Cl. pasteurianum.When enriched iron reducing clostridia were incubated anaerobically under N₂-atmosphere in a sterile, red-colored, lateritic type of soil with glucose, intense gleying occurred within a few days. Microscopic observations indicated the presence of sporeforming bacteria of the Clostridium butyricum type or related species.The biological and chemical mechanism of gley formation is discussed.This research was started at the Institut für Landwirtschaftliche Mikrobiologie, Justus Liebig-Universität, Giessen, Germany.     

Extrem ausgebildete skoliosen der rumpfschwanzregion der wirbelsäule von Gadus

Zusammenfassung Wirbelsäulenanomalien und mißbildungsartige Störungen in der Entwicklung von Einzelwirbeln in leichtem Ausmaß sind bei Fischen nicht selten.Es werden 2 extrem ausgebildete Skoliosen der Wirbelsäule im Bereiche ihrer Rumpf-Schwanzregion an Skeleten von Gadus aus der Ostsee beschrieben und einem völlig gleichsinnigen Präparat von Korschelt auch von Gadus an die Seite gestellt.Anschließend werden die Möglichkeiten der Genese besprochen, ohne daß es gelingt, these völlig zu klären. Vergleichsweise werden 3375 Junglachse von Elterntieren mit Wirbelsäulenverkrümmungen untersucht, um Erblichkeitsmomente zu ermitteln. Ein Anhalt für solche scheint nicht gegeben zu sein.     

Crop rotation and residue management effects on carbon sequestration,nitrogen cycling and productivity of irrigated rice systems

The effects of soil aeration, N fertilizer, and crop residue management on crop performance, soil N supply, organic carbon (C) and nitrogen (N) content were evaluated in two annual double-crop systems for a 2-year period (1994–1995). In the maize-rice (M-R) rotation, maize (Zea mays, L.) was grown in aerated soil in the dry season (DS) followed by rice (Oriza sativa, L.) grown in flooded soil in the wet season (WS). In the continuous rice system (R-R), rice was grown in flooded soil in both the DS and WS. Subplot treatments within cropping-system main plots were N fertilizer rates, including a control without applied N. In the second year, sub-subplot treatments with early or late crop residue incorporation were initiated after the 1995 DS maize or rice crop. Soil N supply and plant N uptake of 1995 WS rice were sensitive to the timing of residue incorporation. Early residue corporation improved the congruence between soil N supply and crop demand although the size of this effect was influenced by the amount and quality of incorporated residue. Grain yields were 13-20% greater with early compared to late residue incorporation in R-R treatments without applied N or with moderate rates of applied N. Although substitution of maize for rice in the DS greatly reduced the amount of time soils remained submerged, the direct effects of crop rotation on plant growth and N uptake in the WS rice crops were small. However, replacement of DS rice by maize caused a reduction in soil C and N sequestration due to a 33–41% increase in the estimated amount of mineralized C and less N input from biological N fixation during the DS maize crop. As a result, there was 11–12% more C sequestration and 5–12% more N accumulation in soils continuously cropped with rice than in the M-R rotation with the greater amounts sequestered in N-fertilized treatments. These results document the capacity of continuous, irrigated rice systems to sequester C and N during relatively short time periods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of Hippurate Hydrolase among Bacillus Species by Thin Layer Chromatography and other Methods

S ummary . The ability of 111 isolates of 33 species of Bacillus to hydrolyze hippurate into benzoic acid and glycine was tested. All, other than the strains classified as B. badius, B. cereus var. mycoides, B. cereus var. thuringiensis, B. macquariensis, B. medusa, B. pacificus, B. psychrosaccharolyticus and some B. macerans and B. polymyxa strains, were positive by thin layer chromatography (TLC). Of the 4 methods compared (hippurate agar, the sulphuric acid method, precipitation with ferric chloride and TLC), only the TLC procedure allowed a rapid and definite detection of the hydrolytic product, benzoic acid. The use of hippurate hydrolase as an additional diagnostic aid in the differentiation of aerobic spore-forming bacilli is discussed. Other characters were used to compare some recently described Bacillus spp. The combination B. cereus subsp. medusa n. subsp. is proposed.     

Effect of NPK on growth and nitrogen fixation of Sesbania rostrata as a green manure for lowland rice (Oryza sativa L.)

The stem-nodulating tropical legume Sesbania rostrata is a promising green manure species for low input rice-farming systems in lowland areas. However, its success as biofertilizer depends on its biomass production and N₂ fixation. Nutrient imbalances and soils low in available nutrients can considerably affect biofertilizer production. Use of mineral N, P, and K fertilizers in growing S. rostrata as biofertilizer for lowland rice was therefore evaluated in pot experiments, and in the fields in Central Luzon, Philippines. Two soils low in Olsen P (3–7.3 mg kg^–1) and exchangeable K (0.05–0.08 meq 100g^-1) were used. Increasing amounts of N (0, 10, 20, 30, and 40 mg kg^-1), P (0, 50, and 100 mg kg^-1), and K (0, 100, 200, and 300 mg kg^-1) were applied to S. rostrata grown in the greenhouse, whereas small amounts of N, P, and K fertilizers (30, 15, and 33 kg ha^-1, respectively) were applied in the field.Mineral N application depressed nodulation and N₂ fixation in roots. It however, stimulated nodulation and N₂ fixation in stems. Applying 30 kg N ha^-1 as urea increased total N accumulation by S. rostrata and yield of the subsequent rice crop (IR64). Applied P and K both stimulated growth, nodulation, and N₂ fixation of S. rostrata. Nitrogen accumulation in P- and K-fertilized S. rostrata was about 40% higher than that in nonfertilized green manure. Thus integration of mineral N, P, and K fertilizers in a green manure-based rice-farming system can considerably improve biofertilizer production and increase rice grain yield.