Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

Isolation of new anaerobic,thermophilic and cellulolytic bacteria-JT strains and their cellulase production

Six microbial strains (JT) of endospore-forming, anaerobic, thermophilic and cellulolytic bacteria were isolated from camel feces, compost, soil and hot spring water in Japan. These strains are gram negative and classified as the genus Clostridium. Strains JT3-1, JT3-2 and JT3-3 can digest starch. All of the strains produce a high activity of extracellular cellulases in cellobiose and cellulose media.Strain JT1 produced 1.36 units/ml of CMCase (endo-β-1,4-d-glucanase, EC 3.2.1.4), 66.2 units/ml of β-glucosidase (ED 3.2.1.21) and 39.9 units/ml of β-xylosidase (EC 3.2.1.37) in 1% cellobiose medium. Strain JT3-3 produced 1.87 units/ml of CMCase, 166.3 units/ml of β-glucosidase and 23.6 units/ml of β-xylosidase in 1% cellulose medium.     

Effects of precursors on serially propagated Digitalis lanata leaf and root cultures

Serially propagated Digitalis lanata leaf and root cultures established from germinated seeds were studied for digoxin production. Leaf cultures were grown and maintained in a medium containing benzyl adenine, and root cultures in the same medium with indoleacetic acid. A consistently high digoxin content, as determined by radio-immunoassay, is present in the leaf culture (9.0 mg % dry wt) and in root culture (1.9 mg % dry wt) as compared to unorganized cells (0.06 mg % dry wt). Leaf liquid cultures grew very rapidly as compared to the root cultures and the unorganized cell suspension cultures. The concentration of digoxin increased in both leaf and root cultures by adding to the medium either sodium glycocholate, cholesteryl acetate, or progesterone. Smilageninacetate increased the digoxin content of root cultures but not that of leaf cultures. Lanosterol and 5β-androstan-3,17-dione did not significantly increase the concentration of digoxin. Deoxycholic acid was toxic to the tissues studied.     

Thermophilic Fermentation of Pig Faeces and Straw by Actinomycetes

The ability of Thermomonospora fusca, Thermomonospora curvata and Pseudonorcardia thermophila to grow on and hydrolyse pig faeces and straw was studied in a 6 d batch culture at 55°C. T. fusca produced the highest levels of cellulase activity (3·3 mg/ml/h) and the greatest cellulose reduction (from 25 to 6% dry wt) in a pig faeces medium (10 g/l). Replacing half the pig faeces with grass straw reduced the cellulose breakdown (29 to 18% dry wt). Increasing the concentration of pig faeces to 30 and 50 g/l caused a decrease in cellulose breakdown. To achieve similar cellulose reductions in straws required NaOH pretreatment. All fermentations resulted in significant increases in digestible protein. The celluloses produced by the strains growing on pig faeces exhibited greatest activity in the pH range 5·9–6·4.     

Glycogen in Methanolobus and Methanococcus

Abstract Ultraviolet light and nitrosoguanidine were used to mutagenize a red pigmented culture of Serratia marcescens , strain EB415, which produced chitinase. After mutagenesis, a stable, non-pigmented mutant designated BL40 was isolated which produced larger colonies and zones of clearing on solid medium containing colloidal chitin.
In liquid medium with colloidal chitin as the sole carbon source both strains grew similarly but BL40 produced 160 units/ml of chitinase compared with 60 units/ml for EB 415, an increase of 167%. When chitin concentration was increased in the medium, chitinase production also increased. Chitinase appeared to be extracellular, since the supernatant from washed, sonicated cells for both strains showed no detectable amount of chitinolytic activity.     

Expression of TPS1 Gene from Saccharomycopsis fibuligera A11 in Saccharomyces sp. W0 Enhances Trehalose Accumulation,Ethanol Tolerance,and Ethanol Production

It has been reported that trehalose plays an important role in stress tolerance in yeasts. Therefore, in order to construct a stably recombinant Saccharomyces sp. W0 with higher ethanol tolerance, the TPS1 gene encoding 6-phosphate-trehalose synthase cloned from Saccharomycopsis fibuligera A11 was ligated into the 18S rDNA integration vector pMIRSC11 and integrated into chromosomal DNA of Saccharomyces sp. W0. The transformant Z8 obtained had the content of 6.23 g of trehalose/100 g of cell dry weight, while Saccharomyces sp. W0 only contained 4.05 g of trehalose/100 g of cell dry weight. The transformant Z8 also had higher ethanol tolerance (cell survival was 25.1 % at 18 ml of ethanol/100 ml of solution) and trehalose-6-phosphate synthase (Tps1) activity (1.3 U/mg) and produced more ethanol (16.4 ml of ethanol/100 ml of medium) than Saccharomyces sp. W0 (cell survival was 12.1 % at 18 ml of ethanol/100 ml of solution, Tps1 activity was 0.8 U/mg and the produced ethanol concentration was 14.2 ml of ethanol/100 ml of medium) under the same conditions. The results show that trehalose indeed can play an important role in ethanol tolerance and ethanol production by Saccharomyces sp. W0.     

Emulsifier of Arthrobacter RAG-1: isolation and emulsifying properties.

The oil-degrading Arthrobacter sp. RAG-1 produced an extracellular nondialyzable emulsifying agent when grown on hexadecane, ethanol, or acetate medium. The emulsifier was prepared by two procedures: (i) heptane extraction of the cell-free culture medium and (ii) precipitation with ammonium sulfate. A convenient assay was developed for measurement of emulsifier concentrations between 3 and 75 micrograms/ml. The rate of emulsion fromation was proportional to both hydrocarbon and emulsifier concentrations. Above pH 6, activity was dependent upon divalent cations; half-maximum activity was obtained in the presence of 1.5 mM Mg2+. With a ratio of gas oil to emulsifier of 50, stable emulsions were formed with average droplet sizes of less than 1 micron. Emulsifier production was parallel to growth on either hydrocarbon or nonhydrocarbon substrates during the exponential phase; however, production continued after growth ceased.     

Emulsifier of Arthrobacter RAG-1: isolation and emulsifying properties.

The oil-degrading Arthrobacter sp. RAG-1 produced an extracellular nondialyzable emulsifying agent when grown on hexadecane, ethanol, or acetate medium. The emulsifier was prepared by two procedures: (i) heptane extraction of the cell-free culture medium and (ii) precipitation with ammonium sulfate. A convenient assay was developed for measurement of emulsifier concentrations between 3 and 75 micrograms/ml. The rate of emulsion fromation was proportional to both hydrocarbon and emulsifier concentrations. Above pH 6, activity was dependent upon divalent cations; half-maximum activity was obtained in the presence of 1.5 mM Mg2+. With a ratio of gas oil to emulsifier of 50, stable emulsions were formed with average droplet sizes of less than 1 micron. Emulsifier production was parallel to growth on either hydrocarbon or nonhydrocarbon substrates during the exponential phase; however, production continued after growth ceased.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Heterocyclic aromatic amine content of selected beef flavors

Previous work has shown that meat extracts contain potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs). Because meat extracts and some beef flavors are produced from similar precursors and processing steps, the beef flavors may also contain HAAs. This study analyzed 24 commercial beef flavors and 2 food-grade beef extracts for creatine and creatinine concentrations, mutagenic activity and HAA concentrations (IQ, MeIQ, MeIQ_x, DiMeIQ_x, Glu-P-1, Glu-P-2 and PhIP). The creatine and creatinine levels of the flavors ranged from 0 to 73 and from 0 to 21 mg/g (dry wt.), respectively. The mutagenic activities of the flavors ranged from 0 to 3200 Salmonella typhimurium TA98 revertants/g (dry wt.). No direct relationship was found between creatine and/or creatinine concentrations and mutagenic activities. However, flavors with high creatine (> 1.5 mg/g) or creatinine (> 2 mg/g) levels exhibited higher mutagenic activities than did flavors with low levels of these compounds. Flavors with high mutagenic activities (> 1500 revertants/g) contained measurable amounts of HAAs. Three flavors contained MeIQ_x (7.2–21.2 ng/g [dry wt.]) and one contained DiMeIQ_x (4.2 ng/g [dry wt.]).     

Characterization of bacterial isolates from industrial wastewater according to probable modes of hexadecane uptake

Bacterial isolates from industrial wastewater were characterized according to probable modes of hexadecane uptake based on data for cell surface hydrophobicity, emulsifying activity, glycoside content and surface tension of cell-free culture medium. The results obtained suggested that both modes of biosurfactant-enhanced hexadecane uptake by bacterial strains take place, direct uptake and alkane transfer. The increase in cell surface hydrophobicity and glycoside production by the strains suggested the existence of biosurfactant-enhanced interfacial uptake of the alkane. Such mechanism is probably predominant for three isolates, Staphylococcus sp. HW-2, Streptococcus sp. HW-9 and Bacillus sp. HW-4. Secreted biosurfactants enhanced mainly alkane emulsification for most hydrophobic isolate Arthrobacter sp. HW-8, and micellar transfer for most hydrophilic isolate Streptococcus sp. HW-5. For other strains (67%) both mechanisms of biosurfactant-enhanced hexadecane uptake probably take place in similar degree, interfacial uptake and alkane emulsification. The results obtained could contribute to clarifying the natural relationships between the members of water ecosystem studied as well as will reveal potential producers of surface active compounds.     

The growth of four species of Azolla as affected by temperature

The growth of 22 strains of Azolla pinnata R. Br., 3 strains of A. filiculoides Lam. and one strain each of A. mexicana Presl and A. caroliniana Willd. was tested separately in liquid culture media kept in controlled, artificial light (30 klux) growth cabinets. Three temperature levels were used: 33°C (37/29°C day/night), 29°C (33/25°C) and 22°C (26/18°C)/ Photoperiod was 12 h a day.For most A. pinnata strains (except three) and an A. mexicana strain the maximum weekly relative growth rate was higher at 33°C than at 22°C, but not for A. filiculoides and A. caroliniana. The highest value of maximum relative growth rate corresponded to 1.9 doubling days and in most strains this occurred in the first week. As the plants grew, the growth rate slowed down more severely at higher temperatures. The maximum biomass was higher at 22°C than at 33°C in all strains. At 22°C, it took 30–50 days to attain maximum biomass and the highest value was 14 g N m^?2 or 320 g dry m^?2 by A. caroliniana, followed by 12 g N m^?2 or 290 g dry wt. m^?2 by one strain of A. filiculoides. At 29°C, the maximum biomass was attained in 20–35 days. The highest value was 6.3 g N m^?2 or 154 g dry wt. m^?2 by A. caroliniana. At 33°C, most A. pinnata strains gave a maximum biomass of less than 4 g N m^?2 after 13–23 days, while some strains grew up to 30 days, resulting in a higher maximum biomass. The highest maximum biomass at 33°C was 5.5 g N m^?2 or 140 g m^?2 dry wt. by A. pinnata from Cheng Mai while the maximum biomass of A. filiculoides and A. caroliniana was much less. Azolla filiculoides requires lower temperature than other species for its growth. Azolla pinnata has the best tolerance to high temperatures among the four species. Azolla mexicana could not be discriminated from A. pinnata in its response to temperature. Azolla caroliniana may keep an intermediate position between A. filiculoides and A. pinnata in temperature response.The formation of ammonia in the medium was examined and it occurred mostly under stationary growth conditions, but, at 33°C, some strains of A. pinnata and A. mexicana released or formed ammonia at 0.3–0.8 μg N ml^?1 per week during their initial exponential growth stage.     

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 μg benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 μg/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 μg/ml, but some hyphae grew on medium amended with 1,000 μg/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 μg/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.     

Biodegradation of indole at high concentration by persolvent fermentation with Pseudomonas sp. ST-200

Pseudomonas sp. strain ST-200 grew on indole as a sole carbon source. The minimal inhibitory concentration of indole was 0.3 mg/ml for ST-200. However, ST-200 grew in a persolvent fermentation system containing a large amount of indole (a medium containing 20% by vol. diphenylmethane and 4 mg/ml indole), because most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1 mg/ml in the presence of diphenylmethane, more than 98% of the indole was consumed after 48 h. Isatic acid (0.4 mg/ml) and isatin (0.03 mg/ml) were produced as the metabolites in the aqueous medium layer. Received: September 12, 1996 / Accepted: January 2, 1997     

Alcohol fermentation in olive oil extraction effluents

Eight culture-collection yeast strains of various species and five newly isolated strains were tested for both growth in olive oil extraction effluents and fermentation of the sugars in the same media. The culture-collection yeast strains did not grow in an effluent containing 2·86% sugar (w/v), 8 g/litre phenolic substances, 4·58 g/litre titratable acidity and pH 4·96, whereas the newly isolated strains of Torulopsis sp. MK-1, Saccharomyces norbensis MC-1, S. oleaceus MC-2 and S. oleaginosus grew well and fermented the sugars. In the medium mentioned above, they produced alcohol in amounts of 1·63 to 1·38%, respectively. None of the yeasts grew in an olive oil extraction effluent vacuum-concentrated to over 13–14% of dry matter. The strain of T. sp. MK-1 showed a hogher stability.     

Kinetics of cell growth and secondary metabolism of a high-tanshinone-producing line of the Ti transformed Salvia miltiorrhiza cells in suspension culture

When cultivated in 6,7-V medium in suspension culture, Salvia miltiorrhiza, transformed with Agrobacterium tumefaciens C58, grew rapidly, reaching about 9.7 g l^–1 dry wt after 12 days. The cell line produced tanshinones: 150 mg cryptotanshinone, 20 mg tanshinone I and 50 mg tanshinone IIA/l and phenolic acids: 530 mg rosmarinic acid and 216 mg lithospermic acid B/l. The phenolic acids were intracellular while about 1/3 of the tanshinones were extracellular. This is the first report of simultaneous production of both phenolic acids and tanshinones in a single culture system.     

Study on Hairy-Root Cultures of Fagopyrum cymosum

Transformed hairy root cultures of Fagopyrum cymosum (Trey.) Meisn. have been established by infecting petioles of shoots cultured in vitro with Agrobacterium rhizogenes. These hairy root cultures grew vigorously in MS hormone-free medium. They showed a 1861-fold increase in fresh weight with in 25 days, and grew faster than cell cultures of F. cymosum (only increased 26.7-fold with in the same days). The cultures were shon to synthesis dimeric procyanidin up to 4.5% (dry wt.) approximating the level of original plant. The growth rates of hairy root cultures up to 149.3 mg dry w-t/L/day in large culture vessels (3 L) and dimeric procyanidin (3.58%, dry wt.) were detected.     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.     

Lactase activity of microorganisms

Sixty-two strains of yeasts, molds and bacteria were screened for lactase (β-D-galactosidase) activity. Strains exhibiting the enzyme activity were evaluated for cell yield as well as enzyme units available per litre of the medium, per g cell dry weight and per mg protein of their cell-free extracts. The molds exhibited lowest enzyme activity but highest cell yields, bacteria produced lowest cell yield and maximum enzyme activity. Cultures exhibiting very high activity among yeasts wereSaccharomyces fragilis (strain 3217) and among bacteriaStreptococcus cremoris (strain H),Lactobacillus bulgaricus (strain RTS and 1373) andLeuconostoc citrovorum (strain 8081).     

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol), whereas when ethanol concentration was changed from 0.1% to 1.6% (wt/vol), the ethanol/glucose ratio for the parent strain changed from 1.6 to 0.6. These data indicate that C. thermohydrosulfuricum strains are tolerant of solvents and that low ethanol tolerance is not a result of disruption of membrane fluidity or glycolytic enzyme activity.