Reversal of the inhibitory effect of surfactants upon germination and growth of a consortium of two strains of Bacillus

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth. Received: 9 July 1998 / Received revision: 26 October 1998 / Accepted: 30 October 1998     

Non-toluene-associated respiration in a Pseudomonas putida 54G biofilm grown on toluene in a flat-plate vapor-phase bioreactor

Non-toluene-associated respiration (NTAR) within a Pseudomonas putida 54G biofilm growing on toluene as sole external carbon source was evaluated using oxygen microelectrodes in a flat-plate vapor-phase biological reactor. Two fluorescent probes, 2,4-diamidino-2-phenylindole and 5-cyano-2,3-ditolyltetrazolium chloride, were used to evaluate the number of total and respiring cells respectively within the biofilm. Biofilm samples were also analyzed for viable and toluene-culturable cells by spread-plating on non-selective and selective media respectively. Fractions of viable stressed, respiring and non-respiring cells within the biofilm were evaluated. The NTAR rate was positively correlated with the fraction of viable stressed and non-respiring cells within the biofilm, which suggested the capability of some cells to grow at the expense of leakage and lysis products coming from injured and dead cells. This effect was more pronounced at higher toluene concentration. Results suggest that NTAR should be incorporated into mathematical models of biofilm reactors degrading volatile organic carbon compounds. Received: 4 January 1997 / Received revision: 20 March 1997 / Accepted: 27 March 1997     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 °C respectively. Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998     

Electrochemical prevention of marine biofouling on a novel titanium-nitride-coated plate formed by radio-frequency arc spraying

We have developed a new method for forming titanium-nitride(TiN)-coated plates using radio-frequency arc spraying (RFAS). A TiN coating formed by RFAS has been used for electrochemical prevention of marine biofouling. X-ray diffraction and X-ray photoelectron spectroscopy indicate that a TiN composite film containing Ti was formed on a polyethylene terephthalate plate surface when Ti was sprayed by RFAS under atmospheric pressure. A cyclic voltammogram (scan rate 20 mV/s) of the TiN formed by RFAS revealed no oxidative and reductive peak currents in the range −0.6 V to 1.2 V against a saturated silver/silver chloride (Ag/AgCl) electrode. When a potential of 1.0 V against Ag/AgCl was applied to the electrode in seawater, no dissolved Ti was detected. Changes in pH and the chlorine concentration were not observed in this range. In all, only 4.5% of the Vibrio alginolyticus cells attached to the electrode survived when a potential of 0.8 V against Ag/AgCl was applied in seawater for 30 min. In field experiments, attachment of the organisms to the TiN electrode was inhibited by applying an alternating potential of 1.0 V and −0.6 V against Ag/AgCl. The TiN film can be formed by RFAS on large and intricately shaped surfaces, and it is a practical electrode for the electrochemical prevention of fouling of various marine structures. Received: 17 April 1998 / Received revision: 5 June 1998 / Accepted: 19 June 1998     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources

When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l⁻¹ sunflower oil fatty acids (technical grade) a yield of 30 g l⁻¹ glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques. Received: 22 June 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

The formation of 1-hydroxymethylnaphthalene and 6-hydroxymethylquinoline by both oxidative and reductive routes in Cunninghamella elegans

Extraction of medium after incubation of the fungus, Cunninghamella elegans, with 0.03% (w/v) 1-methylnaphthalene produced mainly 1-hydroxymethylnaphthalene together with some 1-naphthoic acid and hydroxynaphthoic acid. Higher concentrations of substrate were inhibitory to biotransformation. Similar incubations with 1-naphtoic acid as substrate resulted in reduction of the carboxyl group to give 1-hydroxymethylnaphthalene. When 6-methylquinoline was used, the main product was 6-hydroxymethylquinoline but also some quinoline-6-carboxylic acid and some 6-methylquinoline-N-oxide were identified. In a 2-l fermenter 2.5 g substrate was transformed in 324 h. The 6-hydroxymethylquinoline was also produced by reduction of quinoline-6-carboxylic acid by the organism. Received: 9 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

Stimulatory effect of growth in the presence of alcohols on biotransformation of penicillin G into cephalosporin-type antibiotics by resting cells of Streptomyces clavuligerus NP1

Growth of Streptomyces clavuligerus NP1 in the presence of methanol or ethanol resulted in a marked increase in production of cephalosporin(s) from penicillin G by resting cells. The mycelium produced in alcohol-supplemented medium was fragmented and dispersed as compared with growth in control medium. HPLC analysis showed that at least two products were present in the biotransformation supernatant fluid after 1 h incubation. One of them has been identified as deacetoxycephalosporin G (DAOG). Received: 9 December 1998 / Received revision: 29 March 1999 / Accepted: 16 April 1999     

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 × 10² transformants/μg DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 μF, and 600 Ω. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10¹ transformants/μg DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained. Received: 17 March 1998 / Received revision: 17 August 1998 / Accepted: 26 August 1998     

Adaptation of the filamentous fungus Ashbya gossypii to hyperosmotic stress: different osmoresponse to NaCl and mannitol stress

Osmoadaption mechanisms of the biotechnologically important hemiascomycete Ashbya gossypii were investigated, thereby distinguishing between halo- and osmotolerance by exposure to NaCl and mannitol stress. We studied the growth and ultrastructure of differently treated cells and quantified the intracellular contents of compatible solutes and inorganic ions. Mannitol affected growth of A. gossypii at concentrations above 0.8 M, whereas NaCl inhibited growth at 0.2 M. NaCl-treated cells differed from control cells in having smaller vacuoles, which occupied a smaller part of the cell volume. Glycerol was found to be the predominant compatible solute in A. gossypii; accumulation of inorganic ions could not be detected. Measurement of glycerol uptake under isosmotic conditions as well as upon hyperosmotic stress revealed the existence of a highly active glycerol-uptake system, which, however, was down-regulated under hyperosmotic stress. Investigation of glycerol biosynthesis by measuring glycerol-3-phosphate dehydrogenase activity under hyperosmotic conditions indicated that accumulation of glycerol in A. gossypii is almost solely due to biosynthesis. Received: 13 January 1998 / Received revision: 7 April 1998 / Accepted: 13 April 1998     

Protease-stable integration of Lhcb1 into thylakoid membranes is dependent on chlorophyll b in allelic chlorina-f 2 mutants of barley (Hordeum vulgare L.)

Allelic chlorina-f2 mutants of barley (Hordeum vulgare L.) growing under different light and temperature conditions demonstrated that the chlorophyll b-free chlorina-f 2^{f  2} and chlorina-f 2¹⁰¹ express a stable phenotype. Only 3 out of 10 light-harvesting chlorophyll a/b-binding proteins, Lhca4 (photosystem I), and Lhcb1 and Lhcb6 (photosystem II), required chlorophyll b for accumulation. The other light-harvesting proteins were found in all chlorina-f2 alleles, indicating that the integration pathway of these proteins into mutant thylakoid membranes was not affected. Chlorina-f2 alleles with a thylakoid membrane capable of fullfilling photosynthesis and transport demands, but with various amounts of chlorophyll b: chlorina-f 2¹⁰¹ (chlorophyll b-free), chlorina-f2¹²³ (27% of chlorophyll b compared with the wild type) and chlorina-f 2¹²² (70% chlorophyll b), were chosen to investigate whether chlorophyll b is necessary for the protease-stable insertion of Lhcb l into mutant thylakoid membranes. The Lhcb1 was affected in almost all alleles and was most sensitive to chlorophyll b deficiency. The Lhcb1 antibody confirmed the heterogeneity of the polypeptides of the light-harvesting chlorophyll a/b-binding protein II (LHCII) and detected in wild-type membranes, two protease-resistant, mature forms of Lhcb1 with apparent molecular masses of 28 and 29 kDa. Only one band reacting with the Lhcb1 antibody could be detected in chlorophyll b-free chlorina-f 2  ^{f  2}. It co-migrated with the 29-kDa band, but was completely digested after treatment of the isolated mutant membranes with exogenous protease. This showed that in chlorina-f 2  f  ² the Lhcb1 precursor was processed at one cleavage site only. The resulting 29-kDa Lhcb1 was not provided with chlorophyll b, and, consequently, not properly folded and inserted into the membrane. It remained susceptible to protease and was inconvertable to a 28-kDa form. Received: 23 March 1998 / Accepted: 9 September 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     

Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1

The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined. Received: 12 November 1996 / Received revision: 21 February 1997 / Accepted: 22 February 1997     

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine

A gram-negative, rod-shaped bacterium capable of utilizing l-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed l-asparaginase was detected and it deaminated l-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM l-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on l-fructose, d-galactose, saccharose, or maltose, and in cells cultured on l-asparagine as the sole nitrogen source. The pH and temperature optimum of l-asparaginase was 8.5 and 37–42 °C, respectively. The half-life of the enzyme at 30 °C and 37 °C was 10 and 8 h, respectively. Received: 19 February 1998 / Received last revision: 4 June 1998 / Accepted: 10 July 1998     

Physiology and kinetics of Bifidobacterium bifidum during growth on different sugars

A strain of Bifidobacterium bifidum was grown on different sugars under pH-controlled conditions to estimate some kinetic parameters for growth and product formation. Glucose was the preferred sugar in terms of growth rate and yield, sugar utilisation rate and acetate formation rate, while lactose gave considerably lower values for these parameters. When present in a mixture with glucose, the rate of lactose utilisation was lower than when present on its own. Received: 24 February 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998     

Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Mannitol-enhanced survival of Lactococcus lactis subjected to drying

Survival of Lactococcus lactis subjected to different drying conditions was investigated. Mannitol most remarkably enhanced the survival of dried cells to a level almost equalling that of viable cells [log₁₀ (cfu ml⁻¹) = 9.42] as was found prior to the drying process (log₁₀ = 9.6). In the absence of mannitol, a survival was reduced by a factor of 10⁴. Drying of cells at 20 °C led to higher survival rates than drying at 30 °C. Mannitol enhanced the survival rate at both temperatures, and at both 20 °C and 30 °C the highest reduction in survival occurred when cells were dried at a water activity of 0.76. In the presence of mannitol, differences in survival after drying at different water activities were less pronounced. Rehydration of cells dried in the presence of mannitol resulted in an extended lag phase of 4 h compared to fresh cells. No growth or acidification of the culture medium was observed for 12 h in the case of rehydrated cells dried in the absence of mannitol. It was hypothesized that a radical scavenging activity of mannitol could partly explain these observations. Received: 28 August 1998 / Accepted: 2 October 1998     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

Monitoring nitric oxide from immobilised denitrifying bacteria, Pseudomonas stutzeri, by the use of chemiluminescence

A chemiluminescence detector was used to measure the production of nitric oxide, NO, from the denitrifying bacteria Pseudomonas stutzeri. NO is an intermediate when P. stutzeri converts nitrate into nitrogen gas. The reaction between NO and ozone is selective and sensitive in generating chemiluminescence. Calibrations were made down to 1 nM, with a signal-to-noise ratio of 3. Bacteria were immobilised in alginate beads. Denitrification experiments were made in an anaerobic non-growth medium by adding nitrate to a certain concentration in the reactor. The bacteria were exposed to nitrate in the concentration range 1 pM–5 mM. The lowest concentration to give a measurable NO response was 100 nM. Received: 16 October 1997 / Received revision: 20 January 1998 / Accepted: 24 January 1998