Utilization of immobilized benthic algal species for N and P removal

Laboratory experiments were performed to study the growth rate and phosphorus (P) and nitrogen (N) uptakes of eight benthic microalgae species isolated from different sources of pig manure. Cells immobilized in calcium alginate beads were cultured with three replicates for each species. P removal rates obtained for the unicellular self-aggregating benthic species (Palmellopsis gelatinosa, Chlorosarcinopsis sp., and Macrochloris sp.) were markedly higher than those obtained in previous published experiments. N removal rates were highest for Macrochloris sp., Chlorosarcinopsis sp., and Euglena sp. 2 and comparable to the maximum rates obtained by other authors. Our results show an excellent efficiency of autochthonous benthic species for nutrient removal, especially for P, and call attention to their use for wastewater treatment.     

Construction of an Effective Protein Expression System Using the <Emphasis Type="Italic">tpl</Emphasis> Promoter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis.     

A COMPARISON BETWEEN FLUOROMETRIC AND MASS FRAGMENTOGRAPHIC DETERMINATIONS OF HOMOVANILLIC ACID AND 5-HYDROXYINDOLEACETIC ACID IN HUMAN CEREBROSPINAL FLUID

HVA and 5-HIAA in human cerebrospinal fluid were quantitatively determined by both fluorometry and mass fragmentography. The homovanillic acid values obtained by fluorometry were significantly lower than those obtained by mass fragmentography (P < 0.05). The correlation coefficient between values for HVA obtained by the two methods was high, r= 0.90. For 5-HIAA the concentrations obtained by the two methods were not significantly different while the correlation coefficient was lower, r= 0.55.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Estimation of the D period from residual division after exposure of exponential phase bacteria to chloramphenicol

Summary Values of the D period, between termination of chromosome replication and cell division, were determined from measurements of residual cell division after exposure of exponential phase cultures of Escherichia coli B/r and K12 and of Salmonella typhimurium to chloramphenicol. The results obtained by this method were compared with earlier results for E. coli B/r obtained from measurements of DNA content per cell and were found to be almost identical. For each, values of the D period were independent of growth rate, and the average value of D=26.1±1.2 min obtained by residual division is in good agreement with the value of 25 min obtained earlier. These results indicate that the method of residual division provides a good measure of the duration of the D period. Values of D were also independent of growth rate for each of the other strains.This work supported by the U.S. Atomic Energy Commission.     

Production and properties of three pectinolytic activities produced byAspergillus niger in submerged and solid-state fermentation

Three extracellular pectinases were produced byAspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (K _m values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but theK _m values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number on protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced byA. niger.     

Comparative Chemical Composition of the Essential Oils Obtained by Microwave‐Assisted Hydrodistillation and Hydrodistillation from Agrimonia pilosa Ledeb. Collected in Three Different Regions of China

Conventional hydrodistillation (HD) and microwave‐assisted hydrodistillation (MAHD) were performed to obtain the volatile oils of Agrimonia pilosa Ledeb . harvested in three different regions of China, which were subsequently characterized by GC‐FID and GC/MS analyses. Compared with HD, MAHD was advantageous in terms of energy savings and extraction time (60 vs. 240 min for MAHD and HD, resp.). The chemical composition varied among the different oils obtained, and the variations in the contents of the main constituents of the oils were irregular. Hence, these variations affected both the quantity and composition of the oils. The oil yields (0.15–0.21%) were affected by the method of extraction and the region of harvest, with the maximum amount of oil obtained by MAHD for the plants collected in Hubei (HB) and the minimum yield obtained by HD for the plants from Zhejing (ZJ). Hexadecanoic acid constituted the major compound of the essential oils, with the highest content found in the oil obtained by HD for plants from HB (41.18%) and the lowest one found in the oil obtained by MAHD from plants from ZJ (11.83%). Microwave irradiation did not adversely affect the composition of the essential oils. The findings show that MAHD is a modern, green, and fast technology.     

Production of single-cell protein from enzymatic hydrolyzate of rice straw

Summary The components of rice straw, pretreated with sodium chlorite, cellulose and hemicellulose were solubilized with culture filtrate of Pellicularia filamentosa or Trichoderma reesei. The ratio of glucose to total sugar in the solution obtained from the cellulose component with the culture filtrate of Pellicularia filamentosa was approximately twice that of Trichoderma reesei.Ten yeast strains (Candida utilis, C. tropicalis, C. guilliermondii, C. parapsilosis, Torulopsis xylinus, Trichosporon cutaneum, Debaryomyces hansenii, Rhodotorula glutinis, Saccharomyces fragilis and Saccharomyces cerevisiae) were cultivated as test organisms for single-cell protein (SCP) production on sugar solutions obtained from the straw, cellulose and hemicellulose components, pretreated with the culture filtrate of Pellicularia filamentosa. Sugar consumption, in terms of total sugar and cell yield, of the culture with the sugar solution obtained from pretreated straw were; 70% and 6.8 g/l for Candida tropicalis, 56% and 6.4 g/l for Torulopsis xylinus, 76% and 10.1 g/l for Trichosporon cutaneum, and 74% and 7.6 g/l for Candida guilliermondii. In addition, the highest consumption with respect to total sugar (87%) and the best dry cell yield (15.6 g/l) were observed with the culture of Trichosporon cutaneum using the sugar solution obtained from the hemicellulose component.     

Nucleic acid reduction from yeast activation of intracellular rnase

The study of a heat-shock process for RNA reduction was carried out for different yeast strains. Different results were obtained from each of them. Candida utilis NRRL Y-660 shows its best performance after a 8-s. heat-shock in the presence of 3% NaCl. For commercial baker's yeast Saccharomyces cerevisiae and Kluyveromyces fragilis L-1930, similar results were obtained with only 1% of NaCl. The latter needed longer heat-shock periods. e.g. 15s. to give such an RNA reduction. Biomass recovery ranged from 60 to 75%, being higher for C. utilis and K. fragilis while excessive losses were observed in S. cerevisiae cells. No significant protein deterioration was obtained in the best performance samples. The aminoacid profile appears to be improved in comparison to the starting material in these strains after RNA reduction.     

Application of data consistency tests and new parameter estimation methods to microbial growth on corn dust in batch culture

The microorganism Candida utilis was grown on both filtered and unfiltered substrate obtained from enzymatic hydrolysis of starch in corn dust. For growth on filtered substrate, the average integrated biomass energetic yield value based on biomass-substrate data was η = 0.55 and for growth on unfiltered substrate an average yield value of η = 0.59 was obtained. Material and energy balances showed that the presence of unfiltered corn residue in the media had no significant effect on the yields. Statistical methods were developed and used to obtain best estimates of the growth parameters. Values of the biomass energetic yield corrected for maintenance (η_max = 0.619) and the maintenance coefficient (m_e = 0.043) were obtained for growth on filtered substrate. Values of η_max = 0.741 and m_e = 0.142 were obtained for the growth on unfiltered substrate. The consistency of data and parameter estimates was relatively good for filtered substrate; however, parameter estimates for unfiltered substrate were not consistent. Growth experiments without filtration of the products of starch hydrolysis resulted in protein-enriched products with about 39.73% protein.     

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.     

Use of the rpoB gene as an alternative to the V3 gene for the identification of spoilage and pathogenic bacteria species in milk and milk products

Aim: To evaluate the rpoB gene as an alternative to the V3 gene for the identification of bacterial species in milk and milk products. Methods and Results: DNA obtained from different bacterial species strains was amplified by PCR using rpoB primers. PCR products of each bacterial species were then separated on a DGGE gel. The molecular fingerprints of the bacterial species tested were integrated into a database. The DGGE analysis shows a single band for the rpoB gene amplicons per each bacterial species. Comparison of electrophoretic profiles obtained from V3 16S rDNA amplification with those from this study obtained with rpoB showed that for some bacterial species that co‐migrated after amplification of the V3 region, distinct bands were observed on the gel with the amplification products of the rpoB region. Conclusions: The results obtained in this study show the discriminatory power of the rpoB gene, indicating that it can be used as an alternative to the V3 16S rRNA gene for the identification of bacterial species in milk and milk products. Significance and Impact of the Study: PCR‐DGGE targeting the rpoB gene is a way of discriminating the bacterial species that co‐migrated with the amplification of the V3 gene and so avoids the sequencing of the co‐migrating bands.     

Occurrence of secondary transpositions of Tn5 upon Tn5 mutagenesis inEscherichia coli

When Tn5 insertions were obtained in thehha gene ofEscherichia coli HB101 harboring the hemolytic multicopy plasmid pANN202-312, most of thehha mutants obtained that produced larger amounts of hemolysin than the wild-type cells segregated into 10 percent of clones, which did not further produce hemolysin. We demonstrate here that a secondary transposition of Tn5 intohlyA, the structural gene for hemolysin, was responsible for such phenotype.     

Identification of Cryptococcus neoformans in a routine clinical laboratory

Summary Eight taxonomic tests were compared for their ability to distinguishCryptococcus neoformans from the non-pathogenic species ofCryptococcus. Eight isolates ofCryptococcus were obtained from the American Type Culture Collection and 43 isolates were obtained directly from human and natural sources. The tests which appeared to be most valuable to the routine diagnostic laboratory were growth at 37° C, characteristic growth on Guizotia seed agar and virulence for mice.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

Effect of carbon and nitrogen sources, pH and temperature on in vitro culture of several isolates of Amanita caesarea (Scop.:Fr.) Pers.

Several isolates were obtained from sporocarps of Amanita caesarea (Scop.: Fr.) Pers. associated with Quercus suber and Castanea sativa coming from the southwest of Spain. Culture conditions were optimized for these isolates. The largest radial growth was obtained at pH 6–7, and optimal growth temperature was 24–28°C depending on the isolate. Albumin bovine and nitrate produced the largest patch size diameters, but the greatest mycelium dry weight yields were obtained with ammonium. Mannitol produced the largest radial growth, and mannitol and glucose yielded the biggest mycelium dry weights. Although variations in growth behaviour between isolates were observed, only one internal spacer sequence–restriction fragment length polymorphism type was obtained.     

Spheroplast formation and partial purification of microbodies from hydrocarbon-grown cells ofCladosporium resinae

Summary Cells ofCladosporium resinae form greater numbers of microbodies when grown onn-alkanes than when grown on glucose. To facilitate isolation of microbodies, hydrocarbon-grown cells were spheroplasted. Of four spheroplasting agents and five osmotic supports examined, best results were obtained after a 4-h incubation with Novozym 234 plus chitinase and with 0.8 M sorbitol as osmotic support. Equal numbers of spheroplasts were obtained at pH 5.8 and at pH 7.0. Catalase was used as a marker for microbodies and cytochrome-c oxidase as a marker for mitochondria. Urate oxidase, a second marker for microbodies, was not detected in cell extracts. Microbodies were extremely fragile; of eight spheroplast disruption techniques attempted, the best yield of microbodies was obtained using a Teflon homogenizer for 5 min. Microbodies were partially purified by differential and density gradient centrifugation. Best results were obtained with discontinuous Percoll gradients which yielded a fraction enriched in microbodies and one enriched in mitochondria.     

Preparation of fluorescent labeled nodule bacteria strains of wild legumes for their detection in vivo and in vitro

A series of expression vectors containing TurboGFP and TurboRFP genes of fluorescent proteins under the control of the T5 phage constitutive promoter was created for a vital staining of nodule bacteria. These vectors were either obtained using the broad host range pBBRI replicon for labeling of strains, where a marker gene was expressed from a transformed plasmid, or they were prepared using the pRL765 gfp plasmid for labeling of strains via the introduction of genes of fluorescent proteins into the bacterial chromosome. Transformation was shown to be the most convenient method of transfer of constructions into cells of nodule bacteria, as there exists the possibility of spontaneous plasmid mobilization and, consequently, its transition from cells of labeled strains into other soil bacteria if the mob locus is present in vectors needed for conjugation. Fluorescent labeled strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., and Agrobacterium sp. were prepared using the obtained vector constructions. The suitability of the obtained strains for both in vivo and in vitro experiments was demonstrated.     

Effect ofpH on the formation of Compounds II and III of horseradish peroxidase

The formation of Compounds II and III of horseradish peroxidase from Compound I and potassium ferrocyanide and from Compound II and excess hydrogen peroxide, respectively, was studied as a function ofpH at 25°C and a constant ionic strength of 0.11. The yield of Compound II obtained increases progressively with increase inpH; a mixture of Compounds I and II is produced at acidicpH. Pure Compound III is obtained at allpH values, but the highest yield is obtained atpH values between 6.0 and 7.0. The yield of p-670, formed when Compound III is allowed to stand for 60 min, decreases with increase inpH, while the decay of Compound III also decreases with increase inpH. Therefore p-670 is the decay product of Compound III.