Improvement of pectinase production by interspecific hybrids of Aspergillus strains

Protoplast fusion induced by polyethylene glycol and Ca²⁺, was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 (A13) ade ⁻ and Aspergillus flavipes ATCC-16795 (F7) lys ⁻. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1·0×10⁻². The reversion frequency of the mutation in spores and protoplasts was low and ranged from 2·0 to 4·0×10⁻⁷. Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo-pectinase and pectin-lyase. The highest production was observed in HJ ; maximum activities were 150 and 160% respectively, whereas the exo-pectinase production was similar to the wild-type strain Aspergillus sp. CH-Y-1043. Hybrid HJ showed the greatest growth ; nevertheless, specific endo-pectinase and pectin-lyase activities were higher in all hybrids than those produced by the wild-type strains.     

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

Abstract Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10⁶ cells ml⁻¹). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10⁻⁷ and 10⁻⁴ transconjugants donor⁻¹ in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 10⁵ cells ml⁻¹) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10⁻¹ to 10⁻³) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.     

Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory

The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml⁻¹); B, Artemia (2 nauplii ml⁻¹); C, mixed diet (2 nauplii ml⁻¹ and 3 mg ml⁻¹ microencapsulated diet); and D, microencapsulated diet (3·7 mg ml⁻¹). Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0·376 day⁻¹; regime B, G=0·253 day⁻¹; regime C, G=0·254 day⁻¹; regime D, G=0·162 day⁻¹). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5·6–5·9 mm L_T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8·6–8·7 mm L_T (regimes A, B and C), but only 7·3 mm L_T with regime D. 4·4–6·2 days were required to complete eye migration under the regimes A, B and C, and 18·3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K⁺, Mg²⁺-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent K_m (ATP) and V_max of 0.75 m M and 79 μmol (mg protein)⁻¹ h⁻¹, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent K_m (ATP) of 6.3 m M and V_max of 159 μmol (mg protein)⁻¹ h⁻¹ were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca²⁺ but insensitive to azide and nitrate.     

Studies on catabolite repression in solid state fermentation for biosynthesis of fungal amylases

Catabolite repression by glucose of the biosynthesis of alpha amylase and amyloglucosidase by Aspergillus niger CFTRI 1105 was studied in a solid state fermentation (SSF) and in submerged fermentation (SMF) systems and the results were compared. The addition of glucose did not enhance the production of alpha-amylase and amyloglucosidase in an earlier fermentation system. However, a drastic reduction in alpha-amylase production was observed in submerged fermentation by the addition of 5·0 mg ml⁻¹ glucose and of amyloglucosidase production by 10 mg ml⁻¹ glucose. Glucose concentrations above 50 mg ml⁻¹ completely suppressed the production of both enzymes in the initial hours. In contrast, in the SSF system the repression was negligible, even when the glucose level was raised to 150 mg g⁻¹ wheat bran for both alpha and amyloglucosidase synthesis.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Soluble fractions of Solanum tuberosum enchnce call and pigment production of semi-continous cultures of the microlga Phaeodactylum tricornutum

J. FÁBREGAS, E. MORALES, N. POLANCO, M. PATINO AND A. OTERO. 1996. Soluble fractions of Solanum tuberosum extracted with different methods were tested on semicontinuous mixotrophic cultures of the marine microalga Phaeodactylum tricornutum maintained with renewal rate at 15%. The highest stabilization cell density, 78.2 x 10⁶ cells ml⁻¹, was obtained with an autoclaved non-fermented soluble fraction obtained with distilled water. Highest productivities and carotenoids, 0.9 mg 1⁻¹ d⁻¹, and chlorophyll, 2.9 mg 1⁻¹, d⁻¹, were obtained with a non-autoclaved nonfermented fraction extracted in sea water. The bacterial population associated to the microalgal cultures changed depending on the nutrient availability of each of the potato-soluble fractions.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium-mediated plant transformation

The usefulness of the E. coli codA gene encoding cytosine deaminase as a conditional toxic gene was explored during various stages of plant development and in different Agrobacterium -mediated transformation protocols. To this end, several independent tobacco lines transgenic for codA were isolated and these were tested for their sensitivity to 5-fluorocytosine (5-FC) at different developmental stages. On media supplemented with 5-FC, seedling proliferation was inhibited. Leaves failed completely to regenerate sprouts on 5-FC-containing medium. However, 40% of the shoots regenerated on non-selective medium still formed roots on rooting medium with 5-FC. In all these assays, control plants were unaffected by up to 1 mg m1⁻¹ 5-FC. Transformation of a codA and nptll -harbouring T-DNA to tobacco leaf discs did not result in any regenerant using a combined 5-FC and kanamycin selection, indicating that codA does not behave as a cell-autonomous marker here. Nevertheless, transformation of the same T-DNA to tobacco protoplasts resulted in some enrichment of codA ⁻ nptll ⁺ calluses using the proper combination of 5-FC and kanamycin for selection. Mixing of codA -containing and codA -lacking tobacco protoplasts revealed that the codA gene may behave as a cell autonomous marker under certain, appropriately chosen conditions, which seems to be in paradox with the total absence of escapes in tissue explant transformation. In all these experiments, 250 µg ml⁻¹ 5-FC was found to be the most optimal for selection. Our results suggest that codA can be successfully used as a negative selectable marker in Agrobacterium -mediated gene targeting protocols of tobacco whereby selection at the shoot regeneration level is the most effective.     

Gibberellin enhancement of the enzymic release of Pisum root cell protoplasts

Cultivar differences have been reported in the protoplast yields from Pisum sativum root cortical explants treated with preparations of commercial cellulase and pectinase. The presence of intracellular starch significantly influenced these protoplast yields. The application of gibberellin before or during the enzymic wall-degradation increased the protoplast yields from two of the five cultivars tested. For tissues of'Little Marvel' pea roots, 10 mg 1⁻¹ of gibberellin most effectively increased the release of protoplasts when the hormone preceded the enzyme incubation. One mg 1⁻¹ of gibberellin was most effective at increasing the protoplast release when the tissues were treated with the hormone simultaneously with the wall-degrading enzymes. Mitotic activity was significantly reduced in protoplasts derived from gibberellin-treated tissues.     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Subject index volume 144

Abstract β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the K _m and V _max values have been found to be 1.1 mM and 25.6 μmol min⁻¹(mg protein)⁻¹, respectively. Enzyme activity was inhibited by Hg²⁺, Ag²⁺, and Cu²⁺ at a concentration of 1 × 10⁻³ M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product.     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Kinetics of photoautotrophic growth of Marchantia polymorpha cells in suspension culture

Chlorophyllous, cultured cells of Marchantia polymorpha L. (HYA-2 cell line) grow actively under photoautotrophic (lithotrophic) conditions. The maximum specific growth rate (μ_cell) was 0.64 day⁻¹ and the doubling time was 1.08 days under optimum conditions (165 μmol m⁻² s⁻¹, 1% carbon dioxide enriched atmosphere, 25^°C). The photosynthetic activity was 1.30 μmol CO₂-fixed (10⁶ cells)⁻¹ h⁻¹ [66 μmol (mg chlorophyll)⁻¹ h⁻¹] in the exponential phase. The growth course has two distinct phases, an exponential and a linear one. The exponential phase is observed as long as the population density is sufficiently low (less than 7.9 × 10⁶ cells ml⁻¹), so that practically all individual cells directly receive the full incident light. The effect of light on the specific growth rate is a linear function of photon flux density. Linear growth occurs after the population density is so high that the incident light is almost completely absorbed by the cell suspension. The growth rate is a logarithmic function of photon flux density, in contrast to the specific growth rate, and saturates at high photon flux densities. The conditions of maximum growth, however, are not wellbalanced between cell mass production and cell division. Therefore, the maximum growth does not continue for a long time.     

Simultaneous phototrophic and chemotrophic growth in the purple sulfur bacterium Thiocapsa roseopersicina M1

Abstract The anoxygenic phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in illuminated continuous cultures with thiosulfate as growth limiting substrate. Aeration resulted in completely colorless cells growing chemotrophically, whereafter the conditions were changed to a 23 h oxic/1 h anoxic regime. After 11 volume changes at a dilution rate of 0.031 h⁻¹ (35% of μ_max) a time dependent equilibrium was established. During the 23 h oxic periods bacteriochlorophyll a synthesis (BChl a ) was not observed, whereas during the 1 h anoxic periods synthesis was maximal (i.e. 1.1 μg (mg protein)⁻¹ h⁻¹). As a result the BChl a concentration gradually increased from zero to an average value over 24 h of 1.9 μg (mg protein)⁻¹. Concomitantly, the protein concentration increased from 13.9 mg 1⁻¹ during continuous oxic conditions to 28.8 mg 1⁻¹. For comparison, the protein concentration during fully phototrophic growth at an identical thiosulfate concentration in the inflowing medium was 53.7 mg 1⁻¹. The specific respiration rate was 8 μmol O₂ (mg protein)⁻¹ h⁻¹ during full chemotrophic growth and gradually decreased to 3.5 μmol O₂ (mg protein)⁻¹ h⁻¹ after 11 volume changes at the regime employed. These data show that T. rosepersicina is able to simultaneously utilize light and aerobic respiration of thiosulfate as sources of energy. The ecological relevance of the data is discussed.     

Emission rate of amino acids and ammonia and their role in olfactory preference behaviour of juvenile Arctic charr, Salvelinus alpinus (L.)

The behaviour of juvenile Arctic charr, Salvelinus alpinus (L.) , to an abrupt concentration step of L-amino acids, L-alanine and ammonium chloride was studied by fluviarium technique. The emission rates of these substances were studied. Juvenile Arctic charr emit 8.0 × 10⁻⁴ mol total ammonia-N kg⁻¹ h⁻¹ and 3.3 × I0⁻⁵ mol amino acids kg⁻¹ h⁻¹. In behaviour tests the charr avoided 5.6x 10⁻⁶and 5.6 × 10⁻⁷ M ammonium chloride. The 17 L-amino acid mixture, ranked as observed in the analysis of emission, was avoided at 4.6 × 10⁻⁷ M, while 100 times dilution of this value gave neither avoidance nor attraction. The charr avoided L-alanine tested alone at the concentration of 4.6 × 10 ⁻⁷ M. Anosmic charr showed neither avoidance nor attraction to the mixture of 17 amino acids tested at 4.6 × 10⁻⁷ M. The results indicate that ammonia as well as emitted amino acids are not responsible for the olfactory mediated attraction to conspecific odour shown earlier in Arctic charr. On the contrary, these substances may have a negative effect by reducing the strength of attraction.     

Ammonium and phosphate regenerationby the zooplankton of an Amazon floodplain lake

SUMMARY. 1. Regeneration of ammonium and phosphate by macro-zooplankton (Cladocera. adult copepods. and copepodites) was measured in Lake Calado. an Amazon floodplain lake, Macrozooplanktonabundances ranged between 1×10⁴ and 3×10⁵ individuals m⁻².
2. Phosphate regeneration ranged from 0.2 to 1.3 μ mol PO₄ m⁻² b⁻¹at station 1. located 2 km from the Solimoes River, and from 1.6 to8.3 μ mol PO₄ m⁻² h ⁻¹ at station 3, located 7 km from the SolimoesRiver. Ammonium regeneration at stations 1 and 3 ranged from 1.7 to11.9 and from 13.4 to 77.2 μ mol NH₄ m⁻² h⁻¹. respectively.
3. Zooplankton regenerated ammonium and phosphate at similarrates during rising and falling waier. Regeneration by macrozooplankton was low compared to other tropical lakes and compared to microbesand microzooplankton in Lake Calado.     

Some effects of low pH and calcium on the growth and tissue mineral content of yearling brown trout, Salmo trutta

Yearling brown trout, Salmo trutta , were exposed to low mineral content water (nominal concentrations of 20μmol 1⁻¹ magnesium, 7.7 μmol 1⁻¹ potassium, 44 μmol 1⁻¹ sodium) over a pH range of 4.0–5.2 with ambient calcium concentrations of 2.5–60 μmol 1⁻¹. All fish died at pH 4.0 and 4.2 irrespective of ambient calcium concentration and also at pH 4.4 with only 2–3 μmol 1 ⁻¹ calcium (that is calcium-free water except for that leached from the diet or excreted by the fish). Good growth rates were obtained over the remaining treatments which extended down to pH 4.4 with as little as 7 μmol 1⁻¹ calcium. When starved, weight loss was inversely correlated with pH. Effects on plasma chloride, percentage dry weight and calcium, potassium sodium, and phosphorus contents of skin, muscle and bone tissue were also investigated. These demonstrated pH effects on mineral metabolism in starved fish, but no effects were detected in fed fish.