Prey attraction by luminous larvae of the fungus gnat Orfelia fultoni

Abstract. 1. Bioluminescence in predacious larvae of the fungus gnat Orfelia filtoni attracts potential prey.
2. Transparent traps placed over larvae caught more arthropods than opaque traps put over neighbouring conspecifics.
3. Small Diptera are particularly vulnerable to light lures, while apterous soil arthropods are seemingly unaffected.     

Long-term preservation of anammox bacteria

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.     

Maintenance of biodegradation capacities of aerobic bacteria during long-term preservation

Six strains of aerobic Gram negative bacteria degrading toluene, 2,4-dichlorophenoxyacetate, 2,2-dichloropropionate or 3-chlorobenzoate were freeze-dried and liquid-dried in the presence or absence of a protective agent. Survival and maintenance of the biodegradation capability was checked before and after drying, and after storage of the ampoules for one year at 4° or 25°C. In many cases, stability of the degradation potential was low although viability was high. Survival and stability of all strains was always highest after preservation by liquid drying in the presence of myo-inositol and activated charcoal as protective agents. Losses of biodegradation abilities were highest after freeze-drying using no protective agents. Cells grown on complex medium were less sensitive to drying than cells grown under selective pressure (on mineral medium with a special compound as the sole carbon source). A choice of the most appropriate preservation method and the use of an effective protectant is recommended to avoid genetic alterations, and to maintain biodegradation capacities during long-term preservation.     

Preparation and characterization of medium-chain fatty acid liposomes by lyophilization

In this study, medium-chain fatty acid (MCFA) liposomes were prepared by the film ultrasonic dispersion, modified ethanol injection, and reverse-phase evaporate methods. The results indicated that the liposomes prepared by the thin-film ultrasonic dispersion method had a high entrapment efficiency of 82.7% and a good distribution in size diameters. The MCFA liposomes were freeze-dried and the optimal preparation conditions of freeze-drying were as follows: The cryoprotectants were mannitol and sucrose (1:1 w/w), the hydrated medium was distilled water, and the freeze-drying time was 48 hours. Under these conditions, the freeze-dried MCFA liposomes had a perfect appearance, a small particle size, and high encapsulation efficiency. The mean diameters were 251.1 and 265.3?nm, and the encapsulation efficiencies were 80.5 and 79.2% for freshly prepared and reconstituted liposomes, respectively.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

Variations in the enantioselectivity of salt-activated subtilisin induced by lyophilization

Including excess salt during lyophilization has been shown to increase the activity of freeze-dried subtilisin Carlsberg (SC) in anhydrous media by over 20,000-fold [Ru et al. (1999) Biotechnol Bioeng 63:233-241]. In the present study, salt-activated SC (KCl-SC) showed a 30% enhancement in enantioselectivity compared to the salt-free enzyme in a variety of organic solvents. Activity toward both enantiomers of N-acetyl-phenylalanine methyl ester (APME) increased in tandem by 2-3 orders of magnitude in all solvents, indicating that the mechanism of salt activation is inherent to the enzyme and does not strongly favor one enantiomer over the other. However, activity and enantioselectivity of salt-activated SC could be manipulated through changes in the lyophilization conditions. Variations in lyophilization time, initial KCl concentration, and initial lyophilization volume altered enantioselectivity over 2-fold. The changes in enantioselectivity reflected the activity for the L enantiomer, while the activity toward the D enantiomer was mostly unaffected. The results indicate that the lyophilization time and final water content of the KCl-SC are important determinants of enzyme activity for the L enantiomer, suggesting that the favored reaction is more sensitive to the structural integrity of the salt-activated enzyme.     

Prediction of the preservation of freeze-dried cucumber mosaic virus

A mathematical model to predict the outcome of preserving plant viruses has been developed. The survival of two cucumber mosaic virus strains lyophilized in leaves and in protected plant sap was evaluated using an accelerated storage test at 28, 37 and 45°C. The combinations of 5% (w/v) sorbitol and 7% (w/v) glucose with 3.6% (w/v) dextran 40000 were applied as protecting media. The predicted values to a great extent correspond to the experimental data, achieved after 1, 4 and 7 years of storage.     

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

Mutants of luminous bacteria selected for bioluminescent toxicity tests

Mutants of the luminescent bacterial strain NRRL B-11177 were isolated with pleiotropic hypersensitivity towards hydrophobic antimicrobial agents. SDS-PAGE analyses of outer membrane proteins and lipopolysaccharides revealed that the outer membrane structure of the ahs-mutants was altered. QSAR analysis showed that the inhibitory effect of chloro-substituted phenols on bioluminescence of the ahs-mutants depended on their hydrophobicity. The effect of chlorinated phenols and detergents on bioluminescence was increased in the ahs-mutants. The potential use of these mutants in bioluminescent toxicity tests was discussed.     

Improved preservation of human red blood cells by lyophilization

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.     

Influence of lyophilization, fluidized bed drying, addition of protectants, and storage on the viability of lactic acid bacteria

Aims:  The present study focuses on the impact of two different drying technologies and the influence of protectants on process survival and storage stability of the two lactic acid bacterial strains Enterococcus faecium and Lactobacillus plantarum .
Methods and Results:  After incubation with the protectants glucose, sucrose, trehalose, and maltodextrin the concentrated bacterial suspensions were subjected to fluidized bed drying and lyophilization and subsequently stored at 4, 22, and 35°C for half a year. Lactobacillus plantarum turned out to be more sensitive to both drying methods than Ent. faecium . Without the addition of a protectant cells of both strains suffered higher losses during fluidized bed drying. Elevated storage temperatures correlate with a higher decline of viable bacterial cells.
Conclusions:  Although survival rates varied between the strains, the nonreducing disaccharides revealed overall best protection for both investigated lactic acid bacteria during processing and storage. The addition of protective carbohydrates can prevent the decline in viability during fluidized bed drying.
Significance and Impact of the Study:  The influence of protectants proved to be species specific and therefore needs to be determined on a case-to-case basis. Survival rates, duration, and energy consumption appear to be the crucial parameters to evaluate the economy of production processes for industrial starter cultures.     

冷冻干燥技术在基因工程药物中的应用

冻干技术是基因工程药物制备的常用方法，冻干技术的优点使其正发展成一项产业。介绍了冻干技术的原理、应用、冻干制剂的生产过程，结合工作实际探讨了冻干损伤和保护机理，冻干曲线的确定原则，以及在冻干过程中要注意的重点问题。     

The protective effect of lactose on lyophilization of CNK-20402

The goal of this research was to assess the feasibility of using lyophilization to stabilize an exploratory compound, CNK-20402, with a minimal amount of impurity (CNK-20193) formation. A mixed-level full factorial experimental design was used to screen excipients of glycine, mannitol, lactose monohydrate, and povidone K-12. Cryostage microscopy, powder X-ray diffraction, Karl Fischer titration, HPLC, and water vapor sorption were used to assess the formulations' physicochemical properties and stability. Initial physical characterization from powder X-ray diffraction revealed that the mannitol- and glycine-containing formulations were crystalline with the patterns of the pure excipient, whereas the remaining formulations were amorphous in structure. Chemically, the formulations stored at 50°C for 1 month had 2.36%, 1.05%, 0.81%, 0.79%, and 0.49% CNK-20193 for glycine, mannitol, drug alone, povidone K-12, and lactose formulations, respectively. The formulations containing drug-mannitol, drug alone, and druglactose were selected for accelerated stability study based on statistical analysis. Recovery of CNK-20193 in these formulations was 1.22%, 1.00%, and 0.55%, respectively, when stored at 40°C/75% relative humidity storage conditions for 3 months. Water vapor sorption analysis revealed weight gains of over 7%, 21%, and 24% for the mannitol, lactose, and drug alone formulations, respectively. Testing formulations with different concentrations of lactose by water vapor sorption indicated that CNK-20402 concentrations as low as 10% (wt/wt) could inhibit the recrystallization of lactose. The lactose-containing formulation exhibited the best stability among the formulations tested. The protective mechanism of lactose on the CNK-20402, based on water vapor sorption studies, is believed to be a result of (1) the drug-lactose interaction, and (2) competition between lactose and drug for the residual water in the formulation. Published: September 20, 2005     

Contribution by symbiotically luminous fishes to the occurrence and bioluminescence of luminous bacteria in seawater

Seawater samples from a variety of locations contained viable luminous bacteria, but luminescence was not detectable although the system used to measure light was sensitive enough to measure light from a single, fully induced luminous bacterial cell. When the symbiotically luminous fishCleidopus gloriamaris was placed in a sterile aquarium, plate counts of water samples showed an increase in luminous colony-forming units. Luminescence also increased, decreasing when the fish was removed. Light measurements of water samples from a sterile aquarium containingPhotoblepharon palpebratus, another symbiotically luminous fish, whose bacterial symbionts have not been cultured, showed a similar pattern of increasing light which rapidly decreased upon removal of the fish. These experiments suggest that symbiotically luminous fishes release brightly luminous bacteria from light organs into their environment and may be a source of planktonic luminous bacteria. Although planktonic luminous bacteria are generally not bright when found in seawater, water samples from environments with populations of symbiotically luminous fish may show detectable levels of light.     

Structure and non-enzymatic light emission of two luciferin precursors isolated from the luminous mushroom Panellus stipticus

The chemical structure of two luciferin precursors PS-A and PS-B, isolated from the luminous mushroom Panellus stipticus, were determined as 1-O-decanoylpanal (2) and 1-O-dodecanoylpanal (3), respectively. Both PS-A and PS-B were converted into chemiluminescent luciferins by treatment with 50 mmol/l methylamine in a pH 3.5 buffer solution containing an anionic surfactant Tergitol 4 at 25–35ºC. The luciferins emitted chemiluminescence in a pH 7–8 buffer solution containing a cationic surfactant in the presence of O₂ and O.     

Long-term preservation of yeast cultures by liquid-drying

Various selected strains from about 20 species of yeasts, which are reported to be sensitive to freeze-drying and liquid-drying, were successfully dried directly from the liquid phase without freezing using a simplified liquid-drying method. All tested cultures proved viable and the majority of the tested strains showed good survival rates after drying. However, different survival levels for different yeasts were observed; generally the sensitivity to drying appeared to be strain-specific. After 1 years' storage at 9°C, no further loss in viability was observed. Accelerated storage testing, for 1 week at 45°, resulted in further loss of viability to various degrees. Yeasts that were filamentous, osmotolerant or psychrophilic appeared to be sensitive to liquid-drying and had relatively lower survival levels than the others. Growth and liquid-drying under microaerobic conditions resulted in improved survival. The dried yeast cultures proved stable and no mutation or loss in desirable characters was detected. The method can be used for the drying and long-term preservation of nearly all yeast genera.K.A. Malik and P. Hoffmann are with the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, W-3300 Braunschweig, Germany     

Localisation of luciferase in luminous marine bacteria by gold immunocytochemical labelling

Abstract Bioluminescent bacteria are able to produce light by an oxidative reaction involving the enzyme luciferase. The biochemistry of the reaction has been well documented. It has been postulated from the reaction pathway that the enzyme luciferase is membrane bound. Two species of luminous bacteria, Vibrio fischeri and V. harveyi , were isolated from sea water, fixed and embedded in resin for transmission electron microscopy. A commercially available V. fischeri bacterial luciferase was used to immunise rats for production of an antibody against luciferase. This antibody was coupled with a protein A gold probe and used to immunolabel the sections of bacteria. It was shown that the luciferase was associated with the inner membrane, and to a lesser extent present in the cytoplasm. Our results confirm that the luciferase is predominantly membrane bound.     

Long-term preservation of some Rhodospirillaceae by freeze-drying

Lyophilization of phototropically grown cultures is difficult as it is essential to maintain strict anaerobic conditions or to avoid the exposure of the cultures to light. During a systematic investigation it was observed that several species of Rhodospirillaceae are able to grow heterotrophically in darkness on organic media and are not damaged by air oxygen under such conditions. Based on this character several oxygenic species of Rhodospirillaceae were grown under heterotrophic conditions and were lyophilized using raffinose (5% w/v) along with skim milk (20% w/v) as a protective agent. More than 30 strains from nine species of Rhodospirillaceae were successfully freeze-dried with this method. All tested cultures proved viable and showed 10–100% survival after lyophilization. During 2–3 years of storage at 9°C no further loss in viability was observed. In such lyophilized cultures no loss in photoautotrophy, diazotrophy or other desirable characters like hydrogen production, pigmentation, etc. was detected. The method is not suited to such anoxygenic Rhodospirillaceae which are not able to grown aerobically in darkness.     

Determination of oil oxidation by an aldehyde-requiring mutant of luminous bacteria

A simple, rapid bioluminescence test (BT) for the determination of lipid oxidation is described. The test utilizes an aldehyde-requiring dark mutant of Vibrio harveyi (M42) that emits light in the presence of long chain (C₈-C₁₆) aliphatic aldehydes. The procedure consists of treating the oil or fat with Co²⁺ ion in ethanolic medium at alkaline pH. This treatment facilitates the decomposition of the hydroperoxides into long-chain aldehydes, part of which is used by the bacteria to produce light. The test was evaluated with corn, soybean and safflower oils, and shows excellent correlation with the commonly used peroxide value assay.