Mannose accommodation of <Emphasis Type="Italic">Vigna angularis</Emphasis> cells on solid agar medium involves its possible conversion to sucrose mediated by enhanced phosphomannose isomerase activity

Mannose is an unusable carbon source for many plants. In our study we compared the effects of mannose and sucrose on growth and sucrose levels in azuki bean (Vigna angularis) cells grown in liquid media and in solid media. The suspension cells grew actively in a liquid medium containing 90 mM sucrose but not in that containing 90 mM mannose, where the intracellular sucrose levels were reduced to 20% or less of those in sucrose-grown cells. These results suggested that the limited conversion of mannose to sucrose resulted in cell growth inhibition. When sucrose-grown suspension cells (1 × 10⁵) were transferred onto agar medium containing mannose, they grew little initially, but, after a month lag period, they started to form many callus colonies at a high apparent variation rate (1.3 × 10⁻³). Time-course studies for sugar and enzyme analysis revealed that the mannose-accommodated cells were capable of converting mannose to sucrose, with enhanced phosphomannose isomerase activity. The mannose-accommodated cells actively grew in liquid medium with sucrose but lost their ability to grow with mannose again, suggesting a specific trait of callus culture for mannose utilization. The possible differences in the metabolic activities and other physiological characteristics are discussed between callus and suspension cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Computer-based image analysis for the automated counting and morphological description of microalgae in culture

A largely unexplored area is the application of digital image processing to counting and sizing of microalgal cells from culture. Commercial systems are available, but have not been tested, nor necessarily optimized for high speed counting and sizing of phytoplankton. The present work describes the design, construction, specifications and comparative performance of an inexpensive system optimized for counting and sizing microalgal cells. This system has been tested with cells of the picoplankton to nanoplankton size ranges (1–20 μm). The hardware was a widely available standard microcomputer, an inexpensive video camera and monitor, and a video digitization board (frame grabber). A modifiable menu-driven program (PHYCOUNT) was written and provisions made to make this program available to other workers. The program is constructed such that it can be adapted to a variety of hardware setups Video digitization boards). Comparison of growth curves for microagae revealed there were no significant differences in division rate and cell yield as assessed by the image analysis method compared to manual counts with a hemacytometer. Several hundred cells were counted routinely within 10–15 s, far exceeding the counting rate achieved by hand tally. A variable transect feature allowed sampling every nth pixel and provided a substantial increase in execution speed. More than 1000 counts can be done per day. A protocol for the use of 96-well plates of polyvinyl chloride as counting chambers contributed to the processing of large numbers of samples rapidly. Other routines developed provided subtended area, defined the coordinates of cell perimeter, and derived cell length and width. The calculation of the latter two parameters was usually done off-line as data output is in standard numerical form accessible by other programs. Experience with daily use of the PHYCOUNT program and imaging hardware reveal that the system is reliable for cell counting and sizing. The presence of bacteria in the algal cultures does not affect cell counting or sizing.     

The use of <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis> mutant cell line for removal of cholesterol from milk

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 × 10 ⁶ cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% ± 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.     

RETRACTED ARTICLE: Archaeal <Emphasis Type="Italic">amoA</Emphasis> Genes Outnumber Bacterial <Emphasis Type="Italic">amoA</Emphasis> Genes in Municipal Wastewater Treatment Plants in Bangkok

The contribution of ammonia-oxidizing archaea (AOA) to nitrogen removal in wastewater treatment plants (WWTPs) remains unknown. This study investigated the abundance of archaeal (AOA) and bacterial (ammonia-oxidizing bacteria (AOB)) amoA genes in eight of Bangkok’s municipal WWTPs. AOA amoA genes (3.28 × 10⁷ ± 1.74 × 10⁷–2.23 × 10¹¹ ± 1.92 × 10¹¹ copies l⁻¹ sludge) outnumbered AOB amoA genes in most of the WWTPs even though the plants’ treatment processes, influent and effluent characteristics, removal efficiencies, and operation varied. An estimation of the ammonia-oxidizing activity of AOA and AOB suggests that AOA involved in autotrophic ammonia oxidation in the WWTPs. Statistical analysis shows that the numbers of AOA amoA genes correlated negatively to the ammonium levels in effluent wastewater, while no correlation was found between the AOA amoA gene numbers and the oxygen concentrations in aeration tanks. An analysis of the AOB sequences shows that AOB found in the WWTPs limited to only two AOB clusters which exhibit high or moderate affinity to ammonia. In contrast to AOB, AOA sequences of various clusters were retrieved, and they were previously recovered from a variety of environments, such as thermal and marine environments.     

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

To test the feasibility of CO₂ remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO₂-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO₂ concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO₂ fixation rate of 16.7 g CO₂ l⁻¹ 24 h⁻¹ (or 200.4 g CO₂ m⁻² 24 h⁻¹) could readily be sustained at a light intensity of 2000 μmol m⁻² s⁻¹ at 25 °C, and an ultrahigh cell density of well over 80 g l⁻¹ could be maintained by daily replacing the culture medium. Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998     

An electrochemical method for the measurements of substrate-oxidizing activity of acetic acid bacteria using a carbon-paste electrode modified with immobilized bacteria

In order to measure the substrate-oxidizing activity of intact cells of Acetobacter pasteurianus no. 2, a given amount of the bacterial cells was immobilized on a carbon-paste electrode, and the current at the electrode was measured in a buffer solution. When Fe(CN)³⁻ ₆ was added to the buffer solution, an anodic current was observed at 0.5 V (against Ag/AgCl). Further, when ethanol was added to the solution, the current started to increase to reach a steady-state within 3 min. The electrode had a good response to acetaldehyde and lactic acid as well as ethanol. Culture conditions affected the current response to various substances; the response of the electrode modified with the cells grown in static culture was much higher than that of the electrode with the cells grown in shaking culture, and the electrode with ethanol-grown cells had a high response to ethanol and acetaldehyde compared with that of the electrode with glucose-grown cells. The increase in the amount of the current after the addition of ethanol (ΔI _EtOH) was linearly proportional to the total number of immobilized cells per electrode in the range 1.0 × 10⁴–1.0 × 10⁸ cells. The ΔI _EtOH values were measured with the electrode prepared with a fixed volume of the cell suspensions taken from the culture at 6-h intervals; the dependence of the ΔI _EtOH value on time agreed well with the cell growth measured by colony counting and turbidity in the lag and logarithmic phase. After the logarithmic phase, the value of ΔI _EtOH sharply decreased, resembling to the growth measured by colony counting, rather than by turbidity. Received: 30 October 1998 / Received revision: 2 February 1999 / Accepted: 5 February 1999     

The hemolytic activity of <Emphasis Type="Italic">Karenia selliformis</Emphasis> and two clones of <Emphasis Type="Italic">Karenia brevis</Emphasis> throughout a growth cycle

Blooms of the toxic dinoflagellate, Karenia brevis, occur annually along the Gulf coast of Florida. Other species, like Karenia selliformis, are at times found in association. Hemolytic activity, the ability to lyse red blood cells, of two K. brevis clones (SP3 non-toxic (N-tox) and SP3 super toxic (S-tox)) from the Gulf of Mexico and a single clone of K. selliformis from New Zealand was investigated throughout a growth cycle. Activity is reported as effective concentration (EC₅₀) values, the quantitative measure of hemolysis of human erythrocytes expressed as cell numbers. Both cells and media of K. selliformis cultures consistently produced potent levels of hemolysis (maximum EC₅₀ = 4.88 × 10³ cells) from inoculation until the population declined 35 days later. For SP3 N-tox and S-tox, no hemolytic activity was detectable until day 26 of sampling. The media of both SP3 N-tox and SP3 S-tox cultures consistently contained non-detectable or low levels of hemolysis compared to K. selliformis. Maximum EC₅₀s for the SP3 clones were 1.80 × 10⁶ and 1.97 × 10⁶ cells, respectively. The experimental EC₅₀ values observed represent ecologically relevant cell densities for K. selliformis, but not for the K. brevis clones. In addition, the hemolytic activity of gymnodimine A and various PbTx derivatives was examined in this study. Our findings indicate that the hemolytic capability of these dinoflagellates, especially K. selliformis, represents an additional component of toxicity aside from their already recognized toxins and that this activity may play a larger role than was previously considered. The purpose of this study was to extend the knowledge of the biology and toxicology of species within the genus Karenia.     

Microalgal growth enhancement by levoglucosan isolated from the green seaweed <Emphasis Type="Italic">Monostroma nitidum</Emphasis>

Microalgal growth was enhanced by the addition of levoglucosan to the culture medium. The growth-enhancing compound levoglucosan was isolated from the green seaweed Monostroma nitidum using water extraction, molecular fractionation, DEAE-cellulose column chromatography, and high-performance liquid chromatography. Yield of the compound from seaweed powder was 5 × 10⁻³% (w/w). At 10 mM concentration, levoglucosan enhanced cell growth and the specific growth rate of all feed microalgal species tested (Chaetoceros gracilis, Chlorella ellipsoidea, Dunaliella salina, Isochrysis galbana, Nannochloris oculata, Navicula incerta, Pavlova lutheri, Tetraselmis suecica) in most culture media by approximately 150%. Cellular fatty acid profiles and cell size differed marginally between cultures with and without levoglucosan.     

Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3–2.2 × 10³ CFU g⁻¹ dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g⁻¹ dry soil, equivalent to about 3 × 10⁵ cells g⁻¹ dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3–5 × 10⁷cells g⁻¹ dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment. Received 17 March 1999/ Accepted in revised form 07 April 1999     

High-scale expansion of melanoma-reactive TIL by a polyclonal stimulus: predictability and relation with disease advancement

 The rationale of treating melanoma patients by infusion with tumor-infiltrating leukocytes (TIL) is to perform an adoptive therapy through injection of tumor-specific T cells. Nonetheless, methods currently used for ex vivo TIL expansion have not been evaluated for their efficacy to expand TAA-specific T cells. We have addressed this question here, using a culture method in which high TIL growth was induced by a polyclonal T cell stimulus. Intracellular cytokine assays were performed to measure the proportion of T cells responding to autologous tumor cells among the lymphocytes from lymph node biopsies (TIL) of 26 patients with stage III melanoma. The data show that TIL from 18 of these patients contained detectable amounts of tumor-specific T cells before expansion. Although they decreased somewhat in percent abundance during expansion, they were still present afterwards, ranging from 0.3 to 13.8%. Since a median number of 1.7 × 10¹⁰ TIL was obtained from these patients (starting from 3.6 × 10⁶ TIL), a total amount of tumor-reactive cytokine-secreting TIL of between 2.8 × 10⁶ and 1.12 × 10⁹ was obtained in each case from 18 patients. The TIL populations from 8 patients did not contain tumor-reactive T cells: neither before expansion, nor after expansion. Lack of tumor-reactive TIL only occurs for patients bearing several tumor-invaded lymph nodes (40%), but not for those having a single invaded lymph node. Therefore, high numbers of tumor-reactive T cells can be produced, through a polyclonal TIL stimulation, from most early stage III melanoma patients but from only about half of the patients with a more disseminated disease. For this last group, the possibility of getting tumor-reactive TIL can be predicted by checking the presence of these cells before expansion. Received: 16 November 2000 / Accepted: 18 January 2001     

Production and regeneration of protoplasts from <Emphasis Type="Italic">Grateloupia turuturu</Emphasis> Yamada (Rhodophyta)

Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 10⁷ protoplasts g^-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 10⁶ cells mL^-1 in culture medium and developed into microthalli of a line of two to six cells.     

The influence of hexavalent chromium salt on the population growth,cell morphology,and physiological parameters of the benthic microalga <Emphasis Type="Italic">Attheya ussurensis</Emphasis> Stonik,Orlova, Crawford, 2006 (BACILLARIOPHYTA) in culture

The presence of hexavalent chromium salt in culture medium negatively affected the growth dynamics and physiological parameters of the benthic microalga Attheya ussurensis. After 1 day of exposure to toxicant at concentrations of 2, 4, 7, and 10 mg/l, the cell counts were 10, 7.9, 5.6, and 4.3 × 10³ cells/ml, respectively (versus 13 × 10³ cells/ml in the control). A tendency towards a decrease in cell number remained until the end of the experiments; after 7 days of exposure the cell counts were 133, 102, 11, and 7.5 × 10³ cells/ml (versus 204 × 10³ cells/ml in the control). With increase in potassium bichromate concentration in the culture medium, there was an increase in the ratio of cell height to width and a change in the form of the cell to horseshoe shaped. The contents of chlorophyll a in microalgal cells after 1 day of exposure to 2, 4, 7, and 10 mg/l were 40, 37, 34, and 30 μg/l, respectively (45 μg/l in the control). After 7 days, at chromium salt concentrations of 2 and 4 mg/l, the chlorophyll a content was higher (670 and 647 μg/l) than in the control (605 μg/l); at 7 and 10 mg/l, it significantly decreased to 87 and 65 μg/l, respectively. The contents of carotinoids in microalgal cells after 7 days of exposure to 2 and 4 mg/l were comparable to the control values, while at 7 and 10 mg/l they decreased sharply. The amount of phaeophytin (as a percentage of total chlorophyll a content) increased with increasing potassium bichromate concentration.     

Quantification of Sulfate-reducing Bacteria in Industrial Wastewater,by Real-time Polymerase Chain Reaction (PCR) Using <Emphasis Type="Italic">dsrA</Emphasis> and <Emphasis Type="Italic">apsA</Emphasis> Genes

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5′-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 × 10² and 9.5 × 10⁶ copies/μL and between 1.2 × 10³ and 1.2 × 10⁷ copies/μL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 10⁷–10⁸ targets mL⁻¹) and lower during the winter (about 10⁴–10⁵ targets mL⁻¹). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.     

Seasonal Dynamics and Modeling of a <Emphasis Type="Italic">Vibrio</Emphasis> Community in Coastal Waters of the North Sea

Vibrio species are ubiquitously distributed in marine waters all over the world. High genome plasticity due to frequent mutation, recombination, and lateral gene transfer enables Vibrio to adapt rapidly to environmental changes. The genus Vibrio comprises several human pathogens, which commonly cause outbreaks of severe diarrhea in tropical regions. In recent years, pathogenic Vibrio emerged also in coastal European waters. Little is known about factors driving the proliferation of Vibrio spp. in temperate waters such as the North Sea. In this study a quantification of Vibrio in the North Sea and their response to biotic and abiotic parameters were assessed. Between January and December 2009, Vibrio at Helgoland Roads (North Sea, Germany) were quantified using fluorescence in situ hybridization. Vibrio numbers up to 3.4 × 10⁴ cells × mL⁻¹ (2.2% of total microbial counts) were determined in summer, but their abundance was significantly lower in winter (5 × 10² cells × mL⁻¹). Correlations between Vibrio and nutrients (SiO₂, PO₄ ³⁻, DIN), Secchi depth, temperature, salinity, and chlorophyll a were calculated using Spearman rank analysis. Multiple stepwise regression analysis was carried out to analyze the additive influence of multiple factors on Vibrio. Based on these calculations, we found that high water temperature and low salinity best explained the increase of Vibrio cell numbers. Other environmental parameters, especially nutrients and chlorophyll a, also had an influence. All variables were shown to be subject to the overall seasonal dynamics at Helgoland Roads. Multiple regression models could represent an efficient and reliable tool to predict Vibrio abundances in response to the climate change in European waters.     

Protoplast Preparation from <Emphasis Type="Italic">Laminaria japonica</Emphasis> with Recombinant Alginate Lyase and Cellulase

Functional recombinant abalone alginate lyase (rHdAly) and β-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30°C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35°C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (<1 × 10³ protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 × 10⁶ protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 μm, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.     

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus–insect cell expression system

A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O₂ mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 10⁶ cells ml⁻¹) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 10⁶ cells ml⁻¹ were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell⁻¹, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L⁻¹ of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 10⁶ cells ml⁻¹. Received 09 December 1996/ Accepted in revised form 13 April 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     

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     

Degradation of naphthalene by cells of Pseudomonas sp. strain NGK 1 immobilized in alginate, agar and polyacrylamide

A Pseudomonas sp. strain NGK 1 (NCIM 5120) was immobilized in various matrices, namely, alginate, agar (1.8 × 10¹¹ cfu g⁻¹ beads) and polyacrylamide (1.6 × 10¹¹ cfu g⁻¹ beads). The degradation of naphthalene was studied, by freely suspended cells (4 × 10¹⁰ cfu ml⁻¹) and immobilized cells in batches, with shaken culture and continuous degradation in a packed-bed reactor. Free cells brought about the complete degradation of 25 mmol naphthalene after 3 days of incubation, whereas, a maximum of 30 mmol naphthalene was degraded by the bacteria after 3–4 days of incubation with 50 mmol and 75 mmol naphthalene, and no further degradation was observed even after 15 days of incubation. Alginate-entrapped cells had degraded 25 mmol naphthalene after 3.5 days of incubation, whereas agar- and polyacrylamide-entrapped cells took 2.5 days; 50 mmol naphthalene was completely degraded by the immobilized cells after 6–7 days of incubation. Maximum amounts of 55 mmol, 70 mmol and 67 mmol naphthalene were degraded, from an initial 75 mmol naphthalene, by the alginate-, agar- and polyacrylamide-entrapped cells after 15 days of incubation. When the cell concentrations were doubled, 25 mmol and 50 mmol naphthalene were degraded after 2 and 5.5 days of incubation by the immobilized cells. Complete degradation of 75 mmol naphthalene occurred after 10 days incubation with agar- and polyacrylamide-entrapped␣cells, whereas only 60 mmol naphthalene was degraded by alginate-entrapped cells after 15 days of␣incubation. Further, with 25 mmol naphthalene, alginate-, agar- and polyacrylamide-entrapped cells (1.8 × 10¹¹ cfu g⁻¹ beads) could be reused 18, 12 and 23 times respectively. During continuous degradation in a packed-bed reactor, 80 mmol naphthalene 100 ml⁻¹ h⁻¹ was degraded by alginate- and polyacrylamide-entrapped cells whereas 80 mmol naphthalene 125 ml⁻¹␣h⁻¹ was degraded by agar-entrapped cells. Received: 21 October 1997 / Received revision: 15 January 1998 / Accepted: 18 January 1998     

Sperm cell surface characteristics of Plumbago zeylanica L. in relation to transport in the embryo sac

Myosin associated with the male germ cells of angiosperms interacts with actin, promoting transport of the non-motile generative and later sperm cells in the pollen tube. Myosin localizing on the sperm cell plasma membrane seems negligible in Plumbago, as reflected by the absence of: (i) anti-myosin labeling using immunoelectron microscopy, (ii) sperm motility on actin matrices, and (iii) electrophoretic movement changes after addition of antibody. Sperm cells injected directly into actively streaming Nitella internodal cells, however, follow actin bundles and their movement is sensitive to ATP and Mg²⁺. This may be based on simple charge binding since negatively charged latex beads also migrate on actin, whereas neutral or positively-charged latex beads do not. Sperm cells are negatively charged according to capillary microelectrophoresis, whereas killed sperm cells, which are positively charged do not migrate. The sperm cell that normally fertilizes the egg has a higher calculated charge (8.277 × 10³ esu/cm²) compared with the sperm cell that fuses with the central cell (6.120 × 10³ esu/cm²). Received: 15 December 1998 / Accepted: 21 January 1999