The Influence of Plant Extracts on Phytotoxin Production and Growth Rate of Alternaria helianthi

When liquid cultures of Alternaria helianthi were supplemented with aqueous extracts of leaf tissue of its host plant (sunflower), pronounced effects on both growth and production of the toxin deoxyradicinin were apparent. Very low levels of leaf extract stimulated toxin production but did not significantly affect growth, while higher levels markedly stimulated mycelial growth and at the same time toxin production was greatly suppressed. Leaf extracts of non-host plant species were also stimulatory to phytotoxin biosynthesis.     

3-Epideoxyradicinol and the biosynthesis of deoxyradicinin

A novel phytotoxic compound, in addition to the known phytotoxin deoxyradicinin, has been isolated from Alternaria helianthi and its structure elucidated as 3-epideoxyradicinol. Its epimer is also briefly described. The polyacetate biosynthetic origin of deoxyradicinin has been demonstrated.     

Deoxyradicinin,a novel phytotoxin from Alternaria helianthi

A novel major metabolite which possesses phytotoxic and antifungal properties has been isolated from culture filtrates of Alternaria helianthi and its structure elucidated as deoxyradicinin.     

Effects of rumen fluid collection site on microbial population structure during in vitro fermentation of the different substrates quantified by 16S rRNA hybridisation

Rumen fluid samples from a cow were withdrawn manually from the feed mat (solid phase) or the liquid phase below this mat and incubated in vitro with wheat straw, sorghum hay and a concentrate mixture. From the inoculum and several samples collected during in vitro incubation RNA was extracted to assess microbial population size and structure. RNA content recovered from the solid phase rumen fluid was significantly higher than from the liquid phase. The composition of the microbial population in the solid phase material was characterised by a high proportion of Ruminococci. Neither the proportion of other cell wall degrading organisms (Fibrobacter and Chytridiomycetes) nor the Eukarya and Archaea populations differed between the two sampling sites. Gas production was higher when substrates were incubated with solid phase than with liquid phase rumen fluid regardless of sampling time. However, the higher level of gas production was not accompanied by a corresponding increase in true digestibility. The RNA probes showed that during in vitro incubation with liquid phase rumen fluid, the eukaryotic population was inactive no matter which substrate was used and the activity of methanogens (Archaea) was lower than with solid phase rumen fluid. The population pattern of the cell wall degrading organisms was influenced mainly by the substrate fermented, and to a smaller extent by the inoculum used for in vitro fermentation.     

Mechanism of uptake of liquid hydrocarbons by microorganisms

Growth rates of Candida tropicalis were studied in two different fermentors. One was the ordinary shaker flask containing both the aqueous culture medium and liquid hydrocarbons. The other was a specially designed rotating disk-type fermentor containing only the aqueous culture medium, into which vapors of n-paraffins from C₆ to C₁₈ were supplied continuously without forming the liquid hydrocarbon phase. The specific growth rates of Candida tropicalis in the rotating disk fermentor, under such conditions that supply of hydrocarbon vapor was sufficient, showed good agreement with those in the shaker flask. This seems to indicate that hydrocarbon uptake by Candida tropicals by direct contact with liquid hydrocarbon is negligible.     

Bed expansion characteristics of liquid fluidized particles with attached microbial growth

The bed expansion behavior of liquid fluidized beds of char particles coated with attached microbial growth of denitrifying mixed bacteria was studied experimentally to obtain an expression that relates the bed expansion to the liquid velocity for liquid fluidized beds of composite particles. As for liquid beds of homogeneous spheres, the bed voidage and the liquid velocity v_z for liquid fluidized beds of biomass-coated particles were found to be related as v_z/V_i = ∈ⁿ. The correlations recommended by Richardson and Zaki for homogeneous spheres are found to be satisfactory for the estimation of the constant n, but only under certain conditions for the constant V_i.     

A Thin‐Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Background and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods: A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD₆₀₀ with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD₆₀₀ contained 1.3 ± 0.1 × 10⁹ CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions: Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.     

Comparison of Liquid and Agar-Solidified Defined Media Regarding the Physiological Mechanism by which β-2-Thienylalanine Inhibits Growth of Escherichia,Shigella, and Salmonella Cultures

Growth comparisons were made, using Shigella, Escherichia, and Salmonella cultures, in liquid and agar-solidified defined media containing β-2-thienylalanine (β-2-t). The comparisons were performed to determine the nature of growth inhibition by β-2-t under different physical growth conditions. In a plate assay, with increasing β-2-t mixed into the agar, inhibition of Escherichia and Shigella increased. However, Salmonella cultures were not inhibited even at the highest β-2-t concentrations used. With β-2-t added to liquid cultures, however, dose-response growth relationships were exhibited by all three genera. The differences occurring in β-2-t inhibition between liquid and plate assay conditions were not due to composition of culture plates, time of challenge of cultures with β-2-t, availability of oxygen and associated differences in ratios of volume of media to available surface area, selection of mutants in the plate assay, or to extractable substances from the agar. However, when β-2-t diffusion into the liquid medium was delayed by using agar plug diffusion cultures, a physiological mechanism was demonstrable which largely protected Salmonella cultures, but not Escherichia and Shigella cultures, from growth inhibition.     

细胞焦亡法破碎微生物细胞在合成生物学与代谢工程的应用

【背景】细胞焦亡是一种细胞程序性死亡。在古菌和细菌中,gasdermin同源蛋白（GSDM）能够被特定的活化caspase （protease）酶切,从而激活类似于细胞焦亡的效应,产生细胞破碎效果。【目的】合成生物学、代谢工程和生物制造等应用过程中,细胞破碎是不可或缺的一步。利用细胞焦亡法破碎细胞取代传统的破碎方法,可以简化操作、提高生产效益。【方法】在大肠杆菌（Escherichia coli） BW25113中共表达protease和不同来源的GSDM,选择有明显细胞焦亡效应即来源Runella sp.的GSDM进行蛋白截短改造,使其在诱导表达蛋白截短体GSDMJD后能直接激活细胞焦亡效应。对GSDMJD进行过表达优化,获得可控大肠杆菌细胞焦亡菌株。进一步以重组表达蔗糖磷酸化酶为研究模型,验证本系统应用于细胞破碎释放蛋白的效果。【结果】实现了大肠杆菌中细胞焦亡的人为可控。焦亡菌株在诱导表达焦亡相关蛋白2 h后大肠杆菌细胞破碎死亡,内容物释放。将上述系统和超声法应用于制备蔗糖磷酸化酶粗酶液,细胞焦亡法制备的粗酶液的相对酶活显著高于超声法制备的粗酶液。在制备粗酶液的菌液OD₆₀₀值为2.0时,细胞焦亡法制备的粗酶液相对酶活最高并且相较于超声法制备粗酶液,提高了60%的相对酶活。【结论】细胞焦亡提供了一种更加简单快捷、绿色环保的微生物细胞破碎方式,为合成生物学与代谢工程的发展奠定了基础。     

古代印度三果浆的传入及其影响

三果浆(三勒浆)为源于印度的传统果药剂,由使君子科植物诃子和毛诃子,以及大戟科植物余甘子等三种植物的果实为原料制作而成,是著名的藏药基础方,也是印度阿育吠陀传统药中使用频率最高的代表方剂.三种植物同时录入《中国药典》,余甘子列入我国药食两用植物目录.中古时期,三果浆随着中外文化的频繁交流及丝绸之路的开通传入中国,而今只...     

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and ²⁵²Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17 000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites, Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.     

Dissolved oxygen measurements in pilot- and production-scale novobiocin fermentations

Dissolved oxygen measurements were made in pilot (20 and 250 l.) and production scale (15,000 l.) novobiocin fermentations. Bulk mixing was found to be incomplete in pilot tanks with turbine impellers of D/T = 0.40 (where D is impeller diameter, and T is tank diameter) but appeared homogeneous with impellers of D/T = 0.69. In the former case, the respiration rate was presumably limited by insufficient oxygen supply in areas of poor bulk mixing, whereas, in the latter case, the major resistance was between the bulk of the liquid and the cell (intraclump resistance). Higher agitator speeds decreased the gas–liquid resistance proportionally more than they reduced the liquid-cell resistance. In production fermentors, dissolved oxygen measurements indicated that bulk mixing was complete with each of the three impeller sizes tested (D/T = 0.28, 0.33, and 0.43), but that the respiration rate was again limited, mainly by a resistance between the bulk of the liquid and the cell.     

Water permeation through the lipid bilayer membrane. Test of the liquid hydrocarbon model

According to the liquid hydrocarbon model, the lipid bilayer is viewed simply as a thin slice of bulk hydrocarbon liquid. This allows the water permeability of the bilayer to be calculated from bulk properties. In this paper the prediction of the liquid hydrocarbon model is compared with the known water permeability coefficient of the glycerol monoolein/n-hexadecane bilayer (Fettiplace, R. (1978) Biochim. Biophys. Acta 513, 1–10). As the alkyl chain of glycerol monoolein is equivalent to 8-heptadecene, the water permeability coefficient of 8-heptadecene/n-hexadecane mixtures was measured for temperatures between 20 and 35°C. The mole fraction of n-hexadecane in the bulk liquid was chosen at each temperature to match the known mole fraction of n-hexadecane in the bilayer (White, S. (1976) Nature 262, 421–422). The predicted water permeability coefficient agrees with the measured value at 32°C but is 40% above the measured value at 20°C. The apparent activation energy predicted by the liquid hydrocarbon model is 9.0 ± 0.3 kcal/mol, while the measured value is 14.2 ± 1.0 kcal/mol. The failure of the liquid hydrocarbon model probably results from a different molecular organization of the hydrocarbon chains in the bilayer and in the bulk liquid.     

Evaluation of Epicoccum nigrum for growth, morphology and production of natural colorants in liquid media and on a solid rice medium

Four nonpathogenic and nontoxigenic Epicoccum nigrum strains were evaluated for their growth, morphology and pigment producing ability in three complex and one defined liquid media. Epicoccum nigrum IBT 41028 produced pigments in all the four media tested with a maximum pigment of 3.68 AU at 410 nm in M1 medium (unoptimized) containing 5 g/l yeast autolysate. The color hue of the crude pigment extracts ranged from 74 to 102 exhibiting dark orange to green-yellow color. Pelleted morphology was shown to have a positive influence on the pigment production by E. nigrum strain IBT 41028 in the liquid media, and the use of Bis-tris buffer was found to diminish or reduce the pellet formation. Since Monascus is a well known pigment producer on rice. Pigment producing ability of E. nigrum IBT 41028 was tested on rice and compared to liquid media with Monascus ruber IBT 7904 as control. Though, both genera preferred rice but E. nigrum produced 4.6 folds higher pigment in the liquid unoptimized fermentation medium compared to M. ruber. Solid phase extraction and subsequently HPLC-DAD analysis of the crude pigment extracts showed qualitative as well as quantitative variation in the pigment composition under solid and liquid cultivations.     

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

The ionic liquid 1-alkyl-3-methylimidazolium demonstrates comparable antimicrobial and antibiofilm behavior to a cationic surfactant

Biofilms are problematic in health and industry because they are resistant to various antimicrobial treatments. Ionic liquids are a novel class of low temperature liquid salts consisting of discrete anions and cations, and have attracted considerable interest as safer alternatives to organic solvents. Ionic liquids have interesting antimicrobial properties and some could find use in the development of novel antiseptics, biocides and antifouling agents. The antimicrobial and antibiofilm activity of 1-dodecyl-3-methylimiazolium iodide ([C₁₂MIM]I) was studied using the clinically important bacterial pathogens, Staphylococcus aureus SAV329 and Pseudomonas aeruginosa PAO1. The ionic liquid increased cell membrane permeability in both S. aureus and P. aeruginosa cells and impaired their growth, attachment and biofilm development. The ionic liquid exhibited superior antimicrobial and antibiofilm activity against the Gram-positive S. aureus compared to the Gram-negative P. aeruginosa cells. BacLight? staining and confocal microscope imaging confirmed that the ionic liquid treatment increased the cell membrane permeability of both the Gram-positive and Gram-negative bacteria. In addition, the antimicrobial and antibiofilm properties of [C₁₂MIM]I were similar or superior to those of cetyltrimethylammonium bromide (CTAB), a well-known cationic surfactant. It is concluded that the ionic liquid induced damage to bacterial cells by disrupting cell membrane, leading to inhibition of growth and biofilm formation. Overall, the results indicate that the ionic liquid 1-dodecyl-3-methylimiazolium iodide was effective in preventing S. aureus and P. aeruginosa biofilms and could have applications in the control of bacterial biofilms.     

Isolation of Enterobacteria able to degrade simple aromatic compounds from the wastewater from olive oil extraction

Summary Enterobacteria growing on wastewater from olive oil extraction were selected. Among this microflora, strains of Klebsiella oxytoca and Citrobacter diversus able to degrade simple monomeric aromatic compounds were isolated by enrichment culture of the effluent lacking simple sugars. In this preliminary investigation, the phenolic acids tested on solid and liquid media were gentisic, protocatechuic, p-hydroxybenzoic, benzoic, vanillic and ferulic. It was shown that the biodegradation of an aromatic acid is tightly dependent on both the type and the position of the radical substituted on the aromatic ring. Citrobacter was the most efficient strain in metabolizing ferulic acid in liquid medium at a concentration of 1.5 g/l. The substrate biodegradation yield achieved exceeded 86%.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.