Modeling growth for predicting the contamination level of guava nectar by Candida pelliculosa under different conditions of pH and storage temperature

The combined effects of temperature (2–46°C) and pH (1.55–6.25) on the growth of Candida pelliculosa isolated from guava nectar produced in Cameroon were studied using a turbidity method, ie measurement of optical density at 630 nm. A quadratic polynomial model was constructed to predict the effects and interactions of these two environmental conditions on the maximal optical density obtained (r ² = 0.97). The relation between optical density and population density of C. pelliculosa (CFU ml⁻¹) was also established using an exponential regression (r ² = 0.99). According to the model, maximal growth conditions were 37°C and pH 6.25 for obtaining the maximal optical density of 1.25 corresponding to about 60 × 10⁶ CFU ml⁻¹. A good agreement of the model was found between the predicted values and the observed values of maximal optical density. The model was validated by the experimental values of maximal optical density obtained in the growth of C. pelliculosa in commercial guava nectar (pH 3.15). Received 01 December 1995/ Accepted in revised form 30 August 1996     

Biofilm formation on brass coupons exposed to a cooling system of an oil refinery

Brass coupons (70% Cu 30% Zn) were exposed to a cooling freshwater system of an oil refinery, in order to investigate susceptibility of the metal to biofilm formation. The coupons were fixed on bypasses at points which allowed the circulation of makeup, cooling and return water. The number of aerobic, anaerobic and sulfate-reducing bacteria was determined in both the planktonic and the sessile phases. Maximum bacterial concentrations were detected in the cooling water, corresponding to 2.1 ± 0.1 × 10⁶ CFU ml⁻¹ (planktonic phase) and 1.3 ± 0.2 × 10⁵ CFU cm⁻² (sessile phase) for aerobic bacteria and to 3.2 ± 0.3 × 10⁵ cells ml⁻¹ (planktonic phase) and 6.2 ± 0.7 × 10⁵ cells cm⁻² (sessile phase) for anaerobic bacteria. Sulfate-reducing bacteria (SRB) were observed only in the planktonic phase, being found in greater numbers in the return water. Scanning electron microscopy (SEM) analysis indicated that biofilm formation occurred at the three monitored sites and showed a diversity in cell morphology. Nonetheless, no evidence of corrosion was observed on the brass coupons during the experimental period. Received 22 May 1997/ Accepted in revised form 19 September 1997     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5 × 10⁻⁹ M; binding site II, 1.2 × 10⁻⁸ M; and for the complete promoter/operator region 1 × 10⁻⁸ M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 × 10⁻⁷ M. Received: 18 August 1998 / Accepted: 5 May 1999     

Arctic Sea ice biota: design and evaluation of a mesocosm experiment

A mesocosm experiment (enclosure volume 220 l) was designed such that sea ice inhabited by Arctic Sea ice organisms was formed and maintained under natural conditions at 66°N in Rovaniemi, Finland. The experiment was run from natural freezing in December 1994 to melting in April 1995. The ice was inhabited by diatoms, chlorophyceae, heterotrophic flagellates, ciliates, nematodes and turbellarians. Biomass in the ice, expressed as Chlorophyll a concentration, was 20–110 μg l⁻¹; total cell densities varied from 5 × 10⁶ to 35 × 10⁶ cells l⁻¹. Amongst phototrophic organisms, a succession from a flagellate-dominated community (Chlamydomonas sp.) to a multi-species diatom-dominated community was observed. Typical Arctic species such as Nitzschia frigida and Melosira arctica were present in the ice. Bacterial concentration varied between 2 × 10⁸ and 7 × 10⁸ cells l⁻¹. At least two trophic levels were present in the ice. Received: 3 April 1997 / Accepted: 9 September 1997     

Migratory history and habitat use by New Zealand freshwater eels <Emphasis Type="Italic">Anguilla dieffenbachii</Emphasis> and <Emphasis Type="Italic">A. australis</Emphasis>, as revealed by otolith microchemistry

 The migratory history of Anguilla dieffenbachii and A. australis, collected from a coastal lake of New Zealand, was examined using analysis of strontium (Sr) and calcium (Ca) concentrations. Line analysis of Sr : Ca ratios along the life history transect of each otolith showed a peak (Ca. 16–20 × 10⁻³) between the core and elver mark, which corresponded to the period of their leptocephalus and early glass eel stages in the ocean. The mean Sr : Ca ratios from the elver mark to the otolith edge indicated that eels had different migratory histories, which included freshwater residency in some eels (average Sr : Ca ratios, 1.7 × 10⁻³–2.4 × 10⁻³) but not in others (average Sr : Ca ratios, 3.1 × 10⁻³–6.5 × 10⁻³). These findings suggest that New Zealand freshwater eels have a flexible migration strategy and an ability to adapt to various habitats and salinities. Received: November 25, 2002 / Revised: January 17, 2003 / Accepted: January 17, 2003     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR

Contamination of foods with pathogens such as Escherichia coli O157:H7 and Salmonella is a major concern worldwide and rapid, sensitive, and reliable methods are needed for detection of these organisms. Since these pathogens can contaminate similar foods and other types of samples, a multiplex polymerase chain reduction (PCR) was designed to allow simultaneous detection of both E. coli O157:H7 and Salmonella spp directly from enrichment cultures. Samples of apple cider, beef carcass wash water, ground beef, and bovine feces were inoculated with both E. coli O157:H7 and S. typhimurium at various bacterial levels. Following enrichment culturing for 20–24 h at 37°C in modified EC broth or buffered peptone water both containing novobiocin, the samples were subjected to a DNA extraction technique or to immunomagnetic separation then tested by the multiplex PCR assay. Four pairs of primers were employed in the PCR: primers for amplification of E. coli O157:H7 eaeA, stx _1/2 and plasmid sequences and for amplification of a portion of the Salmonella invA gene. Four fragments of the expected sizes were amplified in a single reaction and visualized following agarose gel electrophoresis in all the samples inoculated with ≤ 1 CFU g⁻¹ or ml⁻¹. Results can be obtained in approximately 30 h. The multiplex PCR is a potentially powerful technique for rapid and sensitive co-detection of both pathogens in foods and other types of samples. Received 28 December 1997/ Accepted in revised form 19 March 1998     

Annual and interannual variability in phytoplankton at a permanent station off Kerguelen Islands, Southern Ocean

From November 1992 to February 1995 a quantitative and qualitative phytoplankton study was conducted at a permanent station (Kerfix) southwest off the Kerguelen Islands, in the vicinity of the Polar Front (50°40′S–68°25′E). Phytoplankton populations are low in this area both during summers and winters. They consist, in order of decreasing cell abundance, of pico- and nanoflagellates (1.5–20 μm), coccolithophorids (<10 μm), diatoms (5–80 μm) and dinoflagellates (6–60 μm). Flagellates form the dominant group throughout the year and attain the highest summer average of 3.0 × 10⁵ cells l⁻¹. Next in abundance year-round are coccolithophorids with the dominant Emiliania huxleyi (highest summer 1992 average 1.9 × 10⁵ cells l⁻¹), diatoms (summer 1992 average 1.0 × 10⁵ cells l⁻¹) and dinoflagellates (average 3.8 × 10⁴ cells l⁻¹). Winter mean numbers of flagellates and picoplankton do not exceed 8.4 × 10⁴ cells l⁻¹; those of the three remaining algal groups together attain 2 × 10⁴ cells l⁻¹. Summer peaks of diatoms and dinoflagellates are mainly due to the larger size species (>20 μm). The latter group contributes most to the total cell carbon biomass throughout the year. Dominant diatoms during summer seasons include: Fragilariopsis kerguelensis, Thalassionema nitzschioides, Chaetoceros dichaeta, C. atlanticus, Pseudonitzschia heimii, and P. barkleyi/lineola. This diatom dominance structure changes from summer to summer with only F. kerguelensis and T. nitzschioides retaining their first and second positions. Any one of the co-dominant species might be absent during some summer period. The variable diatom community structure may be due to southward meandering of the Polar Front bringing “warmer” species from the north, and to the mixing of the water masses in this area. The entire community structure characterized both during summer and winters by the dominance of flagellates can be related to deep mixing (ca. 40–200 m) of the water column as the probable controlling factor. Received: 13 November 1997 / Accepted: 11 May 1998     

Placental Alpha Hemoglobin Stabilizing Protein (AHSP) and recurrent miscarriage

AHSP inhibits cellular production of the reactive oxygen species. Reduced AHSP indicates reduced protection against oxidative stressors. Our objective was to investigate AHSP levels in recurrent miscarriage (RM). Trophoblast was collected from women of 10 weeks gestation: voluntary abortion controls (VA, n = 10); spontaneous first miscarriage with subsequent normal pregnancy (SMSN, n = 15) or with subsequent miscarriage (SMSM, n = 5); RM previously investigated (RMPS, n = 5) or not previously investigated (RM, n = 5). AHSP mRNA and protein were determined using real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. One-way ANOVA was performed to assess statistical significance (p < 0.05). ahsp mRNA levels were maximally reduced in RM and RMPS (8.0 × 10⁻⁶ ± 1.3 and 8.1 × 10⁻⁶ ± 0.7, respectively) compared with SMSN and VA (16.1 × 10⁻⁶ ± 2.3 and 26.1 × 10⁻⁶ ± 2.7, respectively). SMSM showed levels significantly reduced as well (9.0 × 10⁻⁶ ± 2.3). In RM, a reduced defense from oxidative stressors is evident at first miscarriage, identifying women at high risk for subsequent eventful pregnancy. Reduced AHSP may identify women at risk of experiencing further miscarriages. Monica Emanuelli and Monia Cecati contributed equally to this paper.     

Airborne concentrations of bacteria and fungi in Thailand border market

Thailand border market is where the local Thais, Cambodians, Laotians, and Burmeses exchange their goods and culture at the border checkpoints. It is considered to be the source of aerial disease transmission especially for foreigners because it is always very crowded with people from all walks of life. Unhealthy air quality makes this area high risk of spread of airborne diseases. This study assessed airborne concentrations of bacteria and fungi in a border market to improve exposure estimates and develop efficient control strategies to reduce health risk. The density and distribution of airborne bacteria and fungi were investigated in the Chong Chom border market in Surin Province, Thailand. Eighteen air sampling sites were taken from outdoors and various work environments including indoor footpaths, wooden handicraft shops, electronic shops, the secondhand clothing shops, and fruit market areas. Exposed Petri plate method and liquid impinger sampler were used for sampling at the breathing zone, 1.5 m above the floor level, during weekend and holiday. Meteorological factors such as relative humidity, temperature, and light intensity were collected by portable data logger. The relative humidity was 67–73%, and temperature 29–33°C, and light varied between 18 and 270 Lux m⁻². Gram-positive and Gram-negative bacteria were found at a mean value of 10⁴ CFU m⁻³, and airborne fungi of 10³ CFU m⁻³ were recorded. The highest concentration of culturable airborne microorganisms was found along the indoor footpath (9.62 × 10⁴ CFU m⁻³ and 750.00 CFU/plate/h for impingement and sedimentation methods, respectively), the fruit market area (7.86 × 10⁴ CFU m⁻³ and 592.42 CFU/plate/h for impingement and sedimentation methods, respectively), and the secondhand clothing shop (4.59 × 10³ CFU m⁻³ and 335.42 CFU/plate/h for impingement and sedimentation methods, respectively) for Gram-positive bacteria, Gram-negative bacteria, and fungi, respectively. The lowest concentration of Gram-positive bacteria, Gram-negative bacteria, and fungi was found only at the outdoor area at 1.53 × 10⁴ CFU m⁻³, 0.93 × 10⁴ CFU m⁻³ and 0.80 × 10³ CFU m⁻³ by means of impingement method and 136.67 CFU/plate/h, 69.25 CFU/plate/h, and 62.00 CFU/plate/h by means of sedimentation methods for Gram-positive bacteria, Gram-negative bacteria, and fungi, respectively. The most frequently present airborne bacteria were identified as Bacillus, Corynebacteria, Diplococcus, Micrococcus, Acinetobacter, Alcaligenes, Enterobacter, and spore former rods. Acremonium, Aspergillus, Cladosporium, Penicillium, and Sporotrichum were the most frequently found aerosol fungi genera. The distribution of airborne microorganisms correlated with relative humidity and light factors based on principal component analysis. In conclusion, the border market is a potential source of aerial disease transmission and a various hazards of bioaerosols for workers, consumers, sellers, and tourists. The bioaerosol concentration exceeded the standard of occupational exposure limit. Many major indicators of allergenic and toxigenic airborne bacteria and fungi, Acinetobacter, Enterobacter, Pseudomonas, Cladosporium, Alternaria, Aspergillus, and Penicillium, were found in the various market environments.     

Liquid media development for Heterorhabditis bacteriophora : lipid source and concentration

Lipids are important entomopathogenic nematode nutritional components because they are energy reserves and serve as indicators of nematode quality. The composition and concentration of the media lipid component determine bacterial and nematode yields. Heterorhabditis bacteriophora and its symbiont, Photorhabdus luminescens, were cultured in media containing various lipid sources. As lipid concentration increased from 2.5% to 8.0% (w/v), the final yield and productivity [calculated from the number of infective juveniles (IJ)] increased significantly from 2.1 × 10⁵IJ ml⁻¹ to 2.8 × 10⁵IJ ml⁻¹ (P < 0.05) and from 8.9 × 10⁵IJ l⁻¹ day⁻¹ to 11.8 × 10⁵ IJ l⁻¹day⁻¹ (P < 0.05), respectively. The nematode yield coefficient (IJ per gram of media), however, decreased from 2.8 × 10⁶ to 2.2 × 10⁶ (P < 0.05), while recovery increased from 45.3% to 58.0% (P < 0.05). Bacterial cell mass remained constant at 4.6 mg ml⁻¹ with changing lipid content (P > 0.05). The largest nematode yield (2.8 × 10⁵IJ ml⁻¹) was achieved within 8 days, using a medium containing an 8% (w/v) olive and canola oil (50:50 w/v) combination. Moreover, developmental synchrony was achieved in this medium with 96% infective juveniles. In short, lipid sources rich in mono-unsaturated fatty acids and poor in saturated fatty acids produced optimal nematode growth. Received: 1 May 2000 / Received revision: 17 July 2000 / Accepted: 27 July 2000     

Migratory histories of three types of Cottus pollux (small-egg, middle-egg, and large-egg types) as revealed by otolith microchemistry

 Migratory histories of three types of Cottus pollux, the small-egg type (SE type), middle-egg type (ME type), and large-egg type (LE type), were studied by examining strontium (Sr) and calcium (Ca) in their otoliths with wavelength dispersive X-ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed both with ontogenetic development and with salinity of the habitat. Otolith Sr : Ca ratios of LE-type samples and the ME-type samples from the Honmyo River, Kyushu Island, showed consistently low ratios, averaging 1.8 × 10⁻³ and 2.4 × 10⁻³ from the core to the edge, respectively. In contrast, otolith Sr : Ca ratios of SE-type samples and the other four ME-type samples from Hokkaido and Honshu Islands fluctuated strongly along the life history transects in accordance with migration patterns from freshwater to the sea and vice versa. The otolith Sr : Ca ratios of SE-type samples showed low ratios from the core to a point around 15 μm, averaging 1.5 × 10⁻³, and subsequently increased sharply with a high Sr : Ca ratio phase to a point around 400 μm, averaging 5.5 × 10⁻³, and followed again a low ratio phase to the edge with averages of 3.1 × 10⁻³. Similar fluctuation patterns in otolith Sr : Ca ratios were found for the four ME-type samples. These findings clearly demonstrated that otolith Sr : Ca ratios reflected the sculpin's life histories, as being fluvial for the LE type and the Honmyo River ME type and amphidromous for the SE type and the other four populations of ME type. Received: August 1, 2002 / Revised: October 15, 2002 / Accepted: October 28, 2002 Acknowledgments We thank Dr. N. Miyazaki, University of Tokyo, for his kind guidance of our joint research. Thanks are also offered to Drs. H. Sakai, National Fisheries University, Y. Yamazaki, Toyama University, and R. Yokoyama, Hokkaido University, and Mrs. N. Okabe and Y. Suzuki of Yamagata Prefecture for their help in sample collection. This work was partly supported by a Grant-in-Aid (No. 13660171) from the Japan Ministry of Education, Science, Sports and Culture to A. Goto. Correspondence to:Akira Goto     

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.     

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat _GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10⁻⁷ and 4.7 × 10⁻⁷ transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10⁻⁵ transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylorirecA ⁺ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future. Received: 22 April 1997 / Accepted: 4 November 1997     

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K _D = [1.7 ± 0.2] × 10⁻¹⁰ M) and IgG3 (K _D = [1.1 ± 0.2] × 10⁻¹⁰ M) were similar to those of glycosylated rhFcγRI.     

Change in ammonia-oxidizing microorganisms in enriched nitrifying activated sludge

In this study, sludge was taken from a municipal wastewater treatment plant that contained a nearly equal number of archaeal amoA genes (5.70 × 10⁶ ± 3.30 × 10⁵ copies mg sludge⁻¹) to bacterial amoA genes (8.60 × 10⁶ ± 7.64 × 10⁵ copies mg sludge⁻¹) and enriched in three continuous-flow reactors receiving an inorganic medium containing different ammonium concentrations: 2, 10, and 30 mM NH₄⁺–N (28, 140, and 420 mg N l⁻¹). The abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in enriched nitrifying activated sludge (NAS) were monitored at days 60 and 360 of the operation. Early on, between day 0 and day 60 of reactor operation, comparative abundance of AOA amoA genes to AOB amoA genes varied among the reactors depending on the ammonium levels found in the reactors. As compared to the seed sludge, the number of AOA amoA genes was unchanged in the reactor with lower ammonium level (0.06 ± 0.04 mgN l⁻¹), while in the reactors with higher ammonium levels (0.51 ± 0.33 and 0.25 ± 0.10 mgN l⁻¹), the numbers of AOA amoA genes were deteriorated. By day 360, AOA disappeared from the ammonia-oxidizing consortiums in all reactors. The majority of the AOA sequences from all NASs at each sampling period fell into a single AOA cluster, however, suggesting that the ammonium did not affect the AOA communities under this operational condition. This result is contradictory to the case of AOB, where the communities varied significantly among the NASs. AOB with a high affinity for ammonia were present in the reactors with lower ammonium levels, whereas AOB with a low affinity to ammonia existed in the reactors with higher ammonium levels.     

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10⁻⁴ m s⁻¹ (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN₃ or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10⁻⁴ m s⁻¹) to ventral suture (1.32 ± 0.07 × 10⁻⁴ m s⁻¹) and to stylar end (2.53 ± 0.17 × 10⁻⁴ m s⁻¹). There was a positive relationship (r²=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 ± 0.09 × 10⁻⁴ m s⁻¹. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl₃/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ mol⁻¹ for flux and vapour-concentration-based conductance, respectively. Received: 23 March 2000 / Accepted: 28 July 2000     

Comparative analysis of four <Emphasis Type="Italic">β</Emphasis>-galactosidases from <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> NCIMB41171: purification and biochemical characterisation

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.     

Extraction and characterisation of alginate from brown seaweeds (Fucales,Phaeophyceae) collected from Port Dickson,Peninsular Malaysia

Four species of brown seaweeds, namely Sargassum baccularia, Sargassum binderi, Sargassum siliquosum and Turbinaria conoides, harvested from Port Dickson, Negeri Sembilan, Malaysia were analysed for ash content, alginate yield and alginate properties. Seaweeds calcined at 450°C were found to have low amount of non-combustible residue as these were not contaminated by calcareous animals. Alginate was extracted from these seaweeds by two methods: hot and cold. In the hot method, the storing time was 3 h and the processing temperature was 50°C, whilst in the cold method, the sample was stored overnight at room temperature. Higher yield of alginate was obtained by the hot method compared to the cold method, but alginate extracted by the cold method gave higher molecular weight. In the hot method, 49.9% of alginate was extracted from S. siliquosum, followed by T. conoides (41.4%), S. binderi (38.9%) and S. baccularia (26.7%). Alginate extracted from T. conoides has an average molecular weight, M _w, of 8.06 × 10⁵ g mol⁻¹, whereas alginate from S. siliquosum was the lowest in M _w (4.81 × 10⁵ g mol⁻¹) when the extraction was done at room temperature. Alginate extracted from S. baccularia was found to be very heat-sensitive. Its M _w has dropped more than 83%, from 7.52 × 10⁵ to 1.23 × 10⁵ g mol⁻¹, when the extraction temperature was raised. The effect of heat on the extent of depolymerisation of the alginate molecule of the other three brown seaweed species was less significant, with decrease in molecular weight ranging between 13% and 16%.