Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

锰氧化细菌的分离鉴定及其锰氧化特性的分析

利用选择性培养基对锰矿样品进行分离、筛选, 得到一株高效锰氧化细菌(MN1405)。经形态特征、生理生化特征以及16S rRNA 基因序列分析, 将菌株MN1405 鉴定为Arthrobacter echigonensis。在培养条件下, MN1405 对培养基中的锰离子去除率可达93.38%, 且其培养所获得的培养液也具有良好的除锰效果。     

Possible Association of GroES and Antigen 85 Proteins with Heat Resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D_65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D_65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Continuous culture of CHO-K1 cells producing thrombomodulin and estimation of culture conditions

Recombinant Chinese hamster ovary (CHO-K1) cells expressing human soluble thrombomodulin (rsTM) were cultured in a continuous culture system with a fluidized-bed reactor. Cells were grown in a medium containing 1% serum for 10 d, and then cultured in a serum-free medium. The protein production rate increased remarkably in the serum-free culture, with a decrease in the lactate production rate. This suggests that CHO-K1 cells exhibit different physiological characteristics in response to serum removal from the medium, which resulted in a higher rsTM concentration (about 60 mg/l). A procedure for estimating protein productivity was developed using experimental glucose and lactate measurements. In this procedure, cell density was estimated from the glucose consumption rate, and the specific protein (rsTM) production rate was obtained from the ratio of lactate production/glucose consumption (ΔL/ΔG). Since the cell density and protein productivity in repeated batch culture were well estimated, the procedure was applied to continuous culture in a fluidized-bed bioreactor culture. The estimation procedure was also found to be effective in this continuous culture using the models derived from the repeated batch culture.     

Simultaneous nutrient removal and lipid production from pretreated piggery wastewater by Chlorella vulgaris YSW-04

The feasibility of using a microalga Chlorella vulgaris YSW-04 was investigated for removal of nutrients from piggery wastewater effluent. The consequent lipid production by the microalga was also identified and quantitatively determined. The wastewater effluent was diluted to different concentrations ranging from 20 to 80 % of the original using either synthetic media or distilled water. The dilution effect on both lipid production and nutrient removal was evaluated, and growth rate of C. vulgaris was also monitored. Dilution of the wastewater effluent improved microalgal growth, lipid productivity, and nutrient removal. The growth rate of C. vulgaris was increased with decreased concentration of piggery wastewater in the culture media regardless of the diluent type. Lipid production was relatively higher when using synthetic media than using distilled water for dilution of wastewater. The composition of fatty acids accumulated in microalgal biomass was dependent upon both dilution ratio and diluent type. The microalga grown on a 20 % concentration of wastewater effluent diluted with distilled water was more promising for generating high-efficient biodiesel compared to the other culture conditions. The highest removal of inorganic nutrients was also achieved at the same dilution condition. Our results revealed the optimal pretreatment condition for the biodegradation of piggery wastewater with microalgae for subsequent production of high-efficient biodiesel.     

Solid-state culture of Aspergillus niger on support

The growth kinetics of Aspergillus niger on a solid support i.e. sugarcane bagasse, impregnated with a liquid glucose medium, were investigated under different culture conditions. The water activity of the medium, the amount of spore inoculum and the support particle size were shown to be critical factors for mold growth. The elevated rates of growth observed with high substrate concentration media demonstrated the feasibility of the method for culturing filamentous fungi.     

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21°C. Biofilm formation was maximal ca. 20–50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C₆) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer (>C₈) fatty acyl chain.     

Development in vitro of the metacercaria of Bucephaloides gracilescens (Trematoda: Bucephalidae)

The development of Bucephaloides gracilescens metacercariae was studied using a range of cultivation conditions. The most rapid development occurred at 18°C in a medium containing NCTC 135 supplemented with 25% chicken serum, 25% hen egg yolk and 25% hen egg albumen, with a gas phase of air. Under these conditions, shell-protein synthesis was triggered by day 3 in culture; secondary oocytes were apparent in the ovary by day 10; and egg production began by day 14. Survival of worms in media containing chicken serum was twice as long as that achieved with either whiting or angler fish serum. The ingestion of yolk (feeding) appeared to be a necessary prerequisite to development and egg production. The presence of yolk in the culture medium greatly increased the amount of ³H-thymidine incorporated by the reproductive system of freshly excysted metacercariae but had little effect on the uptake and incorporation of tyrosine. The eggs produced in vitro failed to embryonale and were abnormal in appearance, being non-operculate with irregularly thickened shells.     

Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects

The oral toxicity of 5 Photorhabdus spp. strains collected in different regions of Korea was determined in the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, and Culex pipiens pallens. When diet or water containing culture media containing 1 of the 5 different strains was ingested by immature insects, the first instar larvae of both G. mellonella and L. caesar and young larvae of C. pipiens pallens died within 3–5 days after treatment. However, mortality of P. interpunctella neonate larvae was slightly slower and reached 94.4%–100% within 7 days after treatment. The mortality rate of a control group given a diet containing water, the medium without cultured bacteria, or Escherichia coli culture medium was not affected. The mortality rates were 100%, 45.3%, 2.8%, and 0% for Galleria, Lucilia, Plodia, and Culex, respectively, in another control group given a culture medium of Photorhabdus luminescens ssp. laumondii (TT01). In addition, culture media containing Photorhabdus strains significantly inhibited molting of third instar Plodia larvae by as much as 88% 7 days after treatment, whereas molting inhibition was reduced by 0%, 4%, and 20% following treatments with water, E. coli, or TT01 culture media, respectively. Our results suggest that the oral administration of Photorhabdus bacterial medium was highly effective for controlling various immature insects.     

Evaluation of a Most-Probable-Number Technique for the Enumeration of Pseudomonas aeruginosa

A most-probable-number (MPN) technique was evaluated for detecting and enumerating Pseudomonas aeruginosa in water and wastewater. Both the presumptive and confirmatory media, as described in the 13th edition of Standard Methods for the Examination of Water and Wastewater, as well as modifications of these media were included in evaluations. Various samples of water were tested, namely chlorinated tap water, creek water, and influent to a wastewater treatment plant. Modified media repeatedly gave higher estimated MPNs of P. aeruginosa than media listed in Standard Methods. P. aeruginosa was detected and recovered from all creek water and wastewater samples, but not from tap water samples tested. This organism was determined to be present in as large numbers as the fecal coliforms and in even greater quantities than the fecal streptococci in all samples, whenever MPN estimations were determined from those positive tubes containing the modified confirmatory medium.     

Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium

Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log₁₀ reduction of biotin-coated CPMs, and a 2.6-log₁₀ reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log₁₀ reduction, while the uncoated CPMs displayed a 3.7-log₁₀ reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water.     

Biodegradation of glucosinolates in brown mustard seed meal (Brassica juncea) by Aspergillus sp. NR-4201 in liquid and solid-state cultures

Aspergillus sp. NR-4201 was assessed by degrading glucosinolates in brownmustard seed meal (Brassica juncea). A liquid culture of the strain, in a medium derived from the meal, produced total degradation of glucosinolates at 32 h. Under these conditions, the glucosinolate-breakdown product, allylcyanide, was formed inculture filtrates. In a plate culture under sterile conditions, the growth of the strain inheat-treated meal media was shown to be effective at 30 °C with 51% moisture,as determined by the measurement of the colony growth rate. On the laboratory scale,solid-state culture under the same conditions gave rise to total glucosinolate degradationwithin 48 h. In comparison, under non-sterile conditions in either heat-treated or nonheat-treated meal samples, the degradations were complete after 60 and 96 h, respectively.In these cases, growth was associated with some out-growths of contaminating fungi,mainly Rhizopus sp. and Mucor sp. The glucosinolate-breakdown product,allylcyanide, was not detected in the solid-state meal-media culture presumably due toevaporative loss from the fermentation matrix.     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Effects of Inorganic Turbidity on the Phytoplankton of an Amazonian Lake Impacted by Bauxite Tailings

The dumping of bauxite tailings into Batata Lake, an Amazonian clear-water lake, generated high levels of turbidity and caused a serious decrease in phytoplankton densities, which could possibly be the result of a photosynthetic limitation due to light attenuation together with an increase in algal sinking due to the adhesion of clay particles. This study aimed to investigate the sinking process through the addition of different suspended clay concentrations in columns containing Batata lake water. Since no effect of the suspended clays on Batata Lake phytoplankton sinking was observed, it was then evaluated, under laboratory conditions, whether the low conductivity of the Batata Lake water could interfere with the algae-clay aggregation process. Cultures of two algal species known to be capable to aggregate to Batata Lake suspended clays in algal culture medium: Staurodesmus convergens and Phormidium amoenum, were added to both the low conductivity Batata Lake water (14 μS cm^?1) and the high conductivity algal culture media (WC – 300 μS cm^?1 and Z8 – 560 μS cm^?1) together with Batata lake suspended clays. In both algal culture media and Batata lake water the two species had their sinking accelerated due to clay adhesion. It is thus suggested that the decrease in phytoplankton densities recorded in Batata Lake may not be related to an increase in phytoplankton loss rates due to algal-clay aggregation, but rather are a consequence of decreasing growth rates because of light attenuation.     

Growth of submerged mycelia of Aspergillus kawachii in solid-state culture

Submerged mycelial growth of Aspergillus kawachii IFO4308 in solid-state culture (SSC) was studied. From the result of Northern blot analysis, acid-stable α-amylase was found to be produced mainly by the submerged mycelia rather than the aerial mycelia. The submerged mycelia showed better growth in SSC using rice as the solid substrate (koji) than in agar plate culture in spite of low concentrations of dissolved oxygen in koji. Good growth in SSC suggested the existence of an effective oxygen transfer mechanism in koji which governed the mycelial growth. When koji was submerged in water, small bubbles were generated. This phenomenon indicated the formation of vacant spaces in koji during SSC. The submerged mycelia showed better growth in the koji having a larger number of vacant spaces. Considering these facts it was concluded that the vacant spaces participate in effecting an oxygen transfer mechanism in koji as air vents because the diffusivity of oxygen in an air is larger than in koji itself.     

Physiological changes in cultured sorghum cells in response to induced water stress : I. Free proline

Ten varieties of Sorghum bicolor (L.) Moench were grown as callus cultures under conditions of water stress, which was induced by addition of polyethylene glycol (molecular weight 8000) in the medium. Growth and free proline were estimated in the control and water-stressed cultures. In all varieties, proline levels were low in the absence of water stress and the levels increased in response to water stress. However, the magnitude of these increases were not correlated with stress tolerance of the individual varieties in culture. Thus increase in proline seems to be an incidental consequence of stress in vitro and not an adaptive response to combat water stress in sorghum.     

Stimuli for cuticle formation and ecdysis in vitro of the infective larva of Anisakis sp. (Nematoda: Ascaridoidea)

Third-stage larvae of the genus Anisakis from the fish Leionura atun (Trichiuroidei: Perciformes) form a new cuticle and moult in vitro in about 72 h. If the culture medium is Krebs-Ringer under 5% carbon dioxide in air at 37°C, relatively few moult and survival is poor. But more moult and survival is enhanced if worms are incubated in tissue culture medium 199, even if the gas phase is air, although they moult more quickly if it contains 5% carbon dioxide. In both Krebs-Ringer and 199 the benefits of high concentrations of carbon dioxide only accrue if the gas is present during the first 40 h of incubation. Worms do not feed in these media until they have moulted.     

Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture.

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B₁₂-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B₁₂ uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B₁₂. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.     

Promoting effects of organic medium supplements on the micropropagation of promising ornamental Daphne species (Thymelaeaceae)

Three Daphne species (Thymelaeaceae) were propagated in vitro using media enriched with natural ingredients including coconut water, pineapple pulp, arabinogalactan, chitosan, and conditioned medium containing exudates of the green alga Desmodesmus subspicatus. High vigor of proliferative shoots and enhanced rooting efficiency were obtained. The propagation rate for shoot cultures of Daphne caucasica and Daphne tangutica increased significantly when cultured in the presence of 10 ml?L^?1 coconut water or 10 ml?L^?1 pineapple pulp. Addition of 10 ml?L^?1 pineapple pulp, 10 ml?L^?1 coconut water, or 20% conditioned medium to the culture medium stimulated organogenesis in D. caucasica. The percentage of rooted shoots in this difficult-to-root species reached 80% in enriched medium. Daphne jasminea plants rooted efficiently on media without growth regulators but supplemented with 10 ml?L^?1 pineapple pulp or 10 ml?L^?1 coconut water. Plants of D. caucasica and D. jasminea were successfully acclimatized to greenhouse conditions. Biochemical evaluation of pineapple pulp using thin-layer chromatography revealed the absence of natural auxins. However, the low-molecular-weight fraction (<500 Da) obtained via dialysis significantly stimulated rhizogenesis in each species tested.     

The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment

Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent.