Liquoid-Induced Arterial Lesions and the Generalized Shwartzman Reaction

Mural necrotic lesions were produced in renal afferent arteries in rabbits, pigs and blue foxes, by intravenous injections of Liquoid. These lesions were frequently accompanied by thrombosis in the affected arterial segments and invariably by “microthrombosis” in glomerular capillaries. Mural arterial lesions were always present in cases with evident macroscopic necrotic changes of the renal cortex. Necrotic arterial lesions, with thrombosis, were also observed in pulmonary arteries in all the animal species used in these experiments, i.e. rabbit, pig, blue fox, mouse and ferret.     

Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285-296. 1966.-Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO(2) was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus.     

Role of Leukocytes in Blood Coagulation and the Generalized Shwartzman Reaction

IN spite of intensive investigation, many of the factors which initiate blood coagulation and thrombosis remain obscure. The generalized Shwartzman phenomenon, which is due at least in part to intravascular coagulation, is classically obtained by two appropriately spaced, sub-lethal, intravenous doses of endotoxin given to rabbits and is characterized by disseminated thrombi in lungs, kidney and spleen; the characteristic lesion in the kidneys is renal cortical necrosis. Although the Shwartzman phenomenon can be prevented by anticoagulation^{1, 2}, its mechanism remains obscure. Leukocytes have been implicated as the mediators but only indirect evidence is available¹. Leukocytes also possess procoagulant and anticoagulant activity^3–5, the former, however, has always been considered too weak to be physiologically significant or able to cause intensive intravascular clotting with defibrination. We now have evidence that endotoxin given to rabbits may endow their leukocytes with considerable procoagulant activity in vitro, sufficient to produce intravascular clots in various organs when infused to untreated normal rabbits.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

Microbial Production of l-Threonine

The growth of Brevibacterium flavum No. 2247 was inhibited over 90% at a concentration above 1 mg/ml of α-amino-β-hydroxyvaleric acid, a threonine analogue, and the inhibition was reversed by the addition of l-threonine, and to lesser extent by l-leucine, l-isoleucine, l-valine and l-homoserine. l-Methionine stimulated the inhibition. Several mutants resistant to the analogue produced l-threonine in the growing cultures. The percentage of l-threonine producer in the resistant mutants depended on the concentration of the analogue, to which they were resistant. The best producer, strain B-183, was isolated from resistant strains selected on a medium containing 5 mg/ml of the analogue. Mutants resistant to 8 mg/ml of the analogue was derived from strain B-183 by the treatment with mutagen, N-methyl-N’-nitro-N-nitrosoguanidine. Among the mutants obtained, strain BB-82 produced 13.5 g/liter of l-threonine, 30% more than did the parental strain. Among the resistant mutants obtained from Corynebacterium acetoacidophilum No. 410, strain C-553 produced 6.1 g/liter of l-threonine. Several amino acids other than l-threonine were also accumulated, and these accumulations of amino acids were discussed from the view of regulation mechanism of l-threonine biosynthesis.     

Microbial Production of l-Threonine

l-Threonine producing α-amino-β-hydroxyvaleric acid resistant mutants were derived from E. coli K-12 with 3 x 10^-5 frequency. One of mutants, strain β-101, accummulated maximum amount of l-threonine (1. 9 g/liter) in medium. Among isoleucine, methionine and lysine auxotrophs derived from E. coli K-12, only methionine auxotrophs produced l-threonine. In contrast, among isoleucine, methionine and lysine auxotrophs derived from β-101, l-threonine accumulation was generally enhanced in isoleucine auxotrophs. One of isoleucine auxotrophs, strain βI-67, produced maximum amount of l-threonine (4. 7 g/liter). Methionine auxotroph, βM-7, derived from β-101 produced 3.8 g/liter, and βIM-4, methionine auxotroph derived from β1-67, produced 6.1 g/liter, when it was cultured in 3% glucose medium supplemented with 100 μg/ml of l-isoleucine and l-methionine, respectively. These l-threonine productivities of E. coli mutants were discussed with respect to the regulatory mechanisms of threonine biosynthesis. A favourable fermentation medium for l-threonine production by E. coli mutants was established by using strain βM-4.     

Microbial Production of l-Threonine

l-Threonine production by strain BB-69, which was derived from Brevibacterium flavum No. 2247 as a α-amino-β-hydroxyvaleric acid resistant mutant and produced about 12 g/liter of l-threonine, was reduced by the addition of l-lysine or l-methionine in the culture medium. Many of lysine auxotrophs but not methionine auxotrophs derived from strain B–2, which produced about 7 g/liter of l-threonine, produced more l-threonine than the parental strain. Except only one methionine auxotroph (BBM–21), none of lysine and methionine auxotrophs derived from BB–69 produced more l-threonine than the parental strain. Homoserine dehydrogenase of crude extract from strain B–2 was inhibited by l-threonine more strongly than that from BB–69. Strain BBM–21, a methionine auxotroph derived from BB–69, produced about 18 g/liter of l-threonine, 50% more than BB–69, while accumulation of homoserine decreased remarkably as compared with BB–69. l-Threonine production by BBM–21 was increased by the addition of l-homoserine, a precursor of l-threonine, while that by BB–69 was not. No difference was found among BBM–21, BB–69 and No. 2247 in the degree of inhibition of homoserine kinase by l-threonine. l-Threonine production by revertants of BBM–21, that is, mutants which could grow without methionine, were all lower than that of BBM–21. Correlation between l-threonine production and methionine or lysine auxotrophy was discussed.     

The Sequence of Events in the Development of Bilateral Renal Cortical Necrosis Accompanying the Generalized Shwartzman Reaction

The generalized Shwartzman reaction, or Shwartzman-like conditions, were induced in a variety of experimental mammalian species by systemic injections of disintegrated cells of Gram negative bacteria, live Salmonella cholerae-suis or Liquoid. A comparative study of the renal lesions showed that the initial step in the development of bilateral cortical necrosis is stagnation and disintegration of red cells in glomerular capillaries. The glomerular “microthrombi” consist mainly of erythrocytic debris, which frequently has staining properties akin to those of fibrin; even wide-spread glomerular “thrombosis” is not accompanied by obvious destruction of renal parenchyma. A second step is necrotic mural lesions in afferent arteries, with ensuing thrombosis. These vascular lesions lead to the formation of individual infarcts which fuse to form total bilateral cortical necrosis in fulminant cases of the generalized Shwartzman reaction.     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoidosa No.2。研究了培养基组成及培养条件对其细胞生长和油脂积累的影响。结果表明, 最适培养基组成为(g/L)：葡萄糖 20, 甘氨酸 0.08, MgSO4·7H2O 0.4, K2HPO4 1.0, FeSO4·7H2O 0.004; 适宜的培养温度、初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和 650 Lux。在上述优化条件下培养7 d, Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L 和 40.15%提高到6.56 g/L和59.90%, 油脂产量提高了162%。Chlorella pyrenoidosa No.2能以木糖为碳源产油脂, 可望用于以木质纤维素等可再生生物质资源为原料生产油脂。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 不饱和脂肪酸含量达71％左右, 可作为生产生物柴油的原料。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoi-dosa No.2.研究了培养基组成及培养条件对其细胞生长和油脂积累的影响.结果表明,最适培养基组成为(g/L):葡萄糖20,甘氨酸0.08,MgSO4·7H2O 0.4,K2HPO4 1.0,FeSO4·7H2O 0.004;适宜的培养温度,初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和650 Lux.在上述优化条件下培养7 d,Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L和40.15%提高到6.56 g/L和59.90%,油脂产量提高了162%.Chlorella pyrenoidosa No.2能以木糖为碳源产油脂,可望用于以木质纤维素等可再生生物质资源为原料生产油脂.气相色谱分析表明该油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达71%左右,可作为生产生物柴油的原料.     

Microbial Production of Glucose Oxidase

Productivity of extracellular glucose oxidase was examined for various microorganisms and it was found in strains belonging to genus Penicillium except one species of Tallalomyces.

As the best glucose oxidase producer, Penicillium purpurogenum No. 778 was isolated from natural source. This microorganism produced 32,000 units per ml broth of glucose oxidase in a simple medium containing beet molasses, NaNO³ and KH²PO⁴ by submerged culture for 3 days. That value was about 10-times of that of Penicillium amagasakiense which has been known as an excellent glucose oxidase producer.

Culture conditions for glucose oxidase production were examined, which were extremely different among microbial species. In the case of Penicillium chrysogenum AJ 7007 and Penicillium purpurogenum No. 778, the effects of aeration and carbon sources were remarkably different from each other.

Penicillium purpurogenum No. 778 produces catalase sufficiently in a culture broth for glucose oxidase application in food industry.

Glucose oxidase was purified about 25-fold from culture supernatants of Penicillium purpurogenum No. 778, and some properties of the enzyme were examined. The optimum temperature and pH for the activity were 35°C and 5.0, respectively. The enzyme was stable at pH 5.0 to 7.0 when it was incubated at 40°C for 2 hr, while it was stable at temperature lower than 50°C when incubated at pH 5.6 for 15 min. The enzyme was specific for d-glucose and apparent Michaelis constant for d-glucose was 12.5 mm. The enzyme was inhibited by 1 mm of HgCl², CuSO⁴, NaHSO⁴ and phenylhydrazine, but not inhibited by 1 mm of p-hydroxy-mercuribenzoate, EDTA, hydroxylamine and dimedone. Four percents NaCl inhibited the activity about 50%, while the addition of ethanol (from 0 to 16%) increased oxygen uptake more than that expected from the peroxidase activity of catalase.     

The Generalized Shwartzman Reaction in Association With E. Coli Enterotoxemia in a Pig

A pig at the age of approx. 10 weeks died after four days of illness. Distinct necrotic changes were found both in the skin and the cortex of the kidneys. The histological examination revealed fibrinoid thrombi in skin vessels. In the kidneys similar thrombi were observed in capillaries of the glomeruli and in their afferent arterioles and in the interlobular arteries. In these vessels there were also a fibrinoid mural necrosis. These changes were in accordance with those expected to occur in the generalized Shwartzman reaction (GSR). The diagnosis of Escherichia coli enterotoxemia was based on the pathomorphological changes in the alimentary tract. The E. coli enterotoxemia was considered the cause of the GSR-changes.     

Reaction Engineering Aspects of Microbial Mercury Removal

Mercury‐resistant microorganisms are widespread in natural environments and can effectively be used to demercurize Hg(II)‐contaminated wastewaters as was already demonstrated on an industrial scale. The aim of this paper is to find the performance limits with regard to Hg(II) loadings D c_Hg,in (dilution rate × Hg(II) inlet concentration) and residual Hg(II) at the reactor outlet and to provide a reasonable basis for an optimal and safe process design. To this end, comprehensive studies were carried out with different single microbes (natural isolates and a genetically engineered strain) as well as microbial consortia in batch and continuous stirred reactors and fixed beds with microorganisms immobilized as films. The rate of the biotransformation (reduction of inorganically and organically bound Hg(II) to elemental Hg(0)) was found to follow a uniform mechanism with inhibition kinetics (Haldane type). Both reactor types are able to cope with high Hg(II) loadings and yield conversions up to 98 %. The stirred vessel is particularly suited for high c_Hg,in but restricted to low D (D < μ_max), while the fixed bed can be operated at high D, say 10 h^–1, but can only deal with c_Hg,in < 10 mg/L due to the limited Hg(II) tolerance of microorganisms. The loading limitations can be removed by appropriate recycle flows for both reactor types. However, irrespective of reactor type used, the residual Hg at the outlet cannot be reduced below the legal discharge limit (50 μg/L) mainly owing to the adsorption of Hg(II) on biomass. Therefore, a separation step following the reactor is required (sand bed, activated carbon filter). Comparing the reactor types exhibits the superiority of the fixed bed system due to its simpler construction, easier operation and higher cost effectiveness. Furthermore, the fixed bed shows better flexibility and robustness to extreme loadings. This justifies a posteriori the choice of a fixed bed reactor applied in the technical process.     

Restoring Soil Ecosystems and Biomass Production of Arundo donax L. under Microbial Communities-Depleted Soil

In recent years, giant reed (Arundo donax L) has received considerable attention as a promising plant for energy production. Giant reed is able to grow in a range of environments, including wetlands and marginal soils, and has shown promise in phytoremediation efforts. A pot experiment was carried out to investigate the ability of giant reed to restore ecosystems of different soils, including bauxite-derived red mud-amended soil and pure red mud (red mud—a waste generated by the Bayer process in the aluminum industry—is strongly alkaline and has a high salt content and electrical conductivity (EC) dominated by sodium). Samples were exposed to high temperatures, which simulate the effects of bushfires. Selected soil properties that were measured included soil dehydrogenase, alkaline phosphatase, urease and catalase activities, soil organic carbon, soil pH, EC, available soil macronutrients NPK, and above- and below-ground plant biomass yield. The results showed that giant reed reduced EC in all autoclaved soils and red mud-contaminated soils by 24–82 %. Significantly, available N was increased, and a slight increase was recorded for available K. The presence of giant reed enhanced the soils’ enzyme activities to recover in all tested autoclaved soils and red mud-contaminated soils; specifically, dehydrogenase activity increased by 262 and 705 % in non-autoclaved and autoclaved soils, respectively, and urease and catalase activities increased by 591 and 385 % in autoclaved soils, respectively. Total bacterial and fungal counts were higher in autoclaved soils than non-autoclaved soils after cultivating giant reed for 12 weeks. Autoclaved soils enabled higher biomass production for giant reed than non-autoclaved soils. These results demonstrate that giant reed is not only able to survive on soil that has lost its microbial community as a result of heat, but can also yield significant amounts of biomass while assisting recovering soil ecosystems after bushfires.     

Progress in Microbial Carotenoids Production

Carotenoids are versatile isoprenoids pigments, play a vital role in the cellular system, starting from antioxidant to gene regulation. Carotenoids are widely used in food, nutraceuticals, and cosmetics owing to their vitamin A, antioxidant and anticancer activities. The demand of carotenoids in various sectors has triggered the research to explore a commercially viable and environmentally friendly production. This article presents a short review of progress in carotenoids production from microbial platforms.     

Microbial Production of Xylitol from Glucose

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.