Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant

Two plant-growth-promoting bacteria, Azospirillum brasilense Cd and Pseudomonas fluorescens 313, immobilized in 1983 in two types of alginate-bead inoculant (with and without skim-milk supplement) and later dried and stored at ambient temperature for 14 years, were recovered in 1996. The population in each type of bead had decreased, yet significant numbers survived (10⁵–10⁶ cfu/g beads). Population numbers depended on the bead type and the three independent bacterial counting methods: the conventional plate-count method, indirect enzyme-linked immunosorbent assay and the limited-enrichment technique. Both bacterial species retained several of their original physiological features. When inoculated onto wheat plants, both species colonized and produced plant-growth effects equal to those of the contemporary strain from a culture collection or to their own 1983 records. This study showed that bacteria can survive in alginate inoculant over long periods. Received: 1 May 1998 / Received revision: 24 August 1998 / Accepted: 3 September 1998     

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     

Organic halogen removal from chlorinated humic ground water and lake water by nitrifying fluidized-bed biomass characterised by electron microscopy and molecular methods

The dechlorinating and genotoxicity-removing activities of nitrifying fluidized-bed reactor biomass towards chlorinated organic compounds in water were shown at level below 1 ppm. The removal rates of adsorbable organic halogens were 200 μg Cl (g VS day)⁻¹ for chlorinated humic ground water and 50 μyg Cl (g VS day)⁻¹ for chlorinated lake water when studied in batch mode. In a sequenced batch mode the removal rates μg Cl (g VS day)⁻¹] were 2000 from chlorohumus, 1400–1800 from chlorophenols in chlorinated ground water, and 430–720 from chlorohumus in chlorinated lake water. Genotoxicity was removed to a large extent (60%–80%) from the chlorinated waters upon incubation with nitrifying reactor biomass. 2,6-Di-, 2,4,6-tri and 2,3,4,6-tetrachlorophenols competed with chlorinated water organohalogens for dechlorination. The dechlorination of chlorophenols and chlorohumus required no ammonia and was not prevented by inhibitors of ammonia oxidation, nitrapyrin, parathion, sodium diethyldithiocarbamate, or allylthiourea. Electron microscopical inspection of the biomass showed the dominance of clusters of bacteria resembling known nitrifying species, Nitrosomonas, Nitrobacter, and Nitrosospira. This was supported by polymerase chain reaction amplification of the biomass DNA with four different primers, revealing the presence of 16S rDNA sequences assignable to the same species. The most intensive band obtained with the Nitroso4E primer was shown to be closely related to Nitrosomonas europaea by restriction analysis. Received: 27 March 1998 / Received revision: 30 July 1998 / Accepted: 31 July 1998     

Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture

In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (K ^Glc _Lac) was 1.4 ± 0.1 mM and for the dependence of ammonia production on glutamine (K ^Gln _Amm) was 0.25 ± 0.11 mM and 0.10 ± 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This␣metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was␣highly dependent on glucose concentration, the oxygen consumption also increased with the increase in␣glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism. Received: 21 August 1998 / Received revision: 11 November 1998 / Accepted: 17 January 1999     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Analysis of the gene for β-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli

This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with k _cat/K _m values (at 75 °C, pH 5.5) of about 4.1 × 10⁴M⁻¹s⁻¹ and 3.1 × 10⁴M⁻¹s⁻¹ respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology. Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998     

Physiological relationships between auxin, cytokinin, and a peptide growth factor, phytosulfokine-α, in stimulation of asparagus cell proliferation

The aim of this research was to determine whether the production of the mitogenic peptide, phytosulfokine-α (PSK-α), is affected by auxin and/or cytokinin, and whether the expression of the biological activity of PSK-α requires the presence of these plant hormones. We developed a competition enzyme-linked immunosorbent assay system that measures the amount of PSK-α using a polyclonal antibody. In suspension-cultured mesophyll cells of Asparagus officinalis L., the production of PSK-α was first detected after 48 h of culture, prior to the first cell division which was generally observed after 96 h of culture when both 1-napthaleneacetic acid and N⁶-benzyladenine were present in the medium. No significant amount of PSK-α was, however, produced when one of these plant hormones was eliminated from the medium. We also characterized the progression of the cell cycle triggered by PSK-α using a fluorescent dye and microdensitometry. Asparagus mesophyll cells immediately after isolation were arrested in G₀/G₁, and the cell cycle proceeded only when all three factors, 1-naphthaleneacetic acid, N⁶-benzyladenine, and PSK-α, existed in the medium. These results show that the production and the expression of biological activity of PSK-α is closely correlated with the signal transduction pathway mediated by auxin and cytokinin. Received: 26 June 1998 / Accepted: 11 November 1998     

New approaches to control plant parasitic nematodes

Plant parasitic nematodes are a serious threat for crop production worldwide. This review summarizes our understanding of plant nematode interactions and presents new alternatives for nematode control in the field. Breeding for resistance has been a major goal for many important crop species like soybean, potato, tomato and sugar-beet. As a result numerous nematode-resistance genes have been identified, two of which have been cloned recently, Hs1 ^pro-1 from sugar-beet, giving resistance to the beet cyst nematode Heterodera schachtii, and Mi from tomato, giving resistance to the root-knot nematode Meloidogyne incognita. Also artificial resistance genes, coding for nematotoxic proteins or causing rapid death of feeding cells, have been elucidated. In the future, genetic engineering of nematode resistance will become more and more important for plant breeding. Transformation techniques will allow genes to be quickly introduced into susceptible breeding lines and then combined with each other to produce plant varieties with durable resistance. Received: 26 August 1998 / Received revision: 16 December 1998 / Accepted: 21 December 1998     

Pullulan content of the ethanol precipitate from fermented agro-industrial wastes

Ethanol-precipitated substances after fermentation of various agro-industrial wastes by Aureobasidium pullulans were examined for their pullulan content. Grape skin pulp extract, starch waste, olive oil waste effluents and molasses served as substrates for the fermentation. A glucose-based defined medium was used for comparison purposes. Samples were analysed by an enzyme-coupled assay method and by high-performance anion-exchange chromatography with pulsed amperometric detection after enzymic hydrolysis with pullulanase. Fermentation of grape skin pulp extract gave 22.3 g l⁻¹ ethanol precipitate, which was relatively pure pullulan (97.4% w/w) as assessed by the coupled-enzyme assay. Hydrolysed starch gave only 12.9 g l⁻¹ ethanol precipitate, which increased to 30.8 g l⁻¹ when the medium was supplemented with NH₄NO₃ and K₂HPO₄; this again was relatively pure pullulan (88.6% w/w). Molasses and olive oil wastes produced heterogeneous ethanol-precipitated substances containing small amounts of pullulan, even when supplemented with nitrogen and phosphate. Overall, grape skin pulp should be considered as the best substrate for pullulan production. Starch waste requires several hydrolyis steps to provide a usable carbon source, which reduces its economic attraction as an industrial process. Received: 24 October 1997 / Received revision: 10 February 1998 / Accepted: 15 February 1998     

The turnover of cell surface proteins of carrot protoplasts

The covalent modification of cell surface proteins with N-hydroxysuccinimide esters of biotin was used to develop a strategy for following the turnover of proteins on the surface of carrot (Daucus carota L.) protoplasts. A biotinylation/internalisation assay was established which enabled the turnover of cell surface proteins to be examined by biochemical and immunocytochemical techniques. The detection of biotinylated proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that a variety of proteins on the surface of the protoplasts were covalently modified. Immunolocalisation of biotinylated proteins in protoplasts directly after their derivatisation, demonstrated that the proteins were initially restricted to the cell surface. Incubation of biotinylated protoplasts at 25 °C for 1 h resulted in the detection of biotin-labelled proteins on the cell surface and intracellularly. A small proportion of these proteins was associated with coated pits, the Golgi apparatus and vacuolar compartments. Biochemical analysis of internalised proteins revealed that a polypeptide of approximate M_r 100 000 was internalised by the protoplasts. Immunolabelling of a biotinylated protein of M_r 100 000 by an antibody raised against an isoform of a tobacco plasma-membrane H⁺-ATPase, strongly suggests that the plasma-membrane H⁺-ATPase is internalised by carrot protoplasts. The implications of these results are discussed within the context of endocytosis in plants. Received: 13 July 1998 / Accepted: 11 November 1998     

Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-l sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l⁻¹ h⁻¹. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH. Received: 16 November 1998 / Received revision: 28 March 1999 / Accepted: 9 April 1999     

Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67

Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoelectric point at pH 9.75. The results of amino acid composition analyses showed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °C respectively. The enzyme was significantly activated by the addition of 1 mM Mn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺ ions in the reaction mixture, and its thermal stability was significantly increased by Ca²⁺ ion. Received: 31 August 1998 / Received last revision: 1 December 1998 / Accepted: 20 December 1998     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R. capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures. Received: 16 March 1998 / Accepted: 28 July 1998     

Fungal laccase grafts acrylamide onto lignin in presence of peroxides

Laccase (EC 1.10.3.2) from the white-rot basidomycete Trametes versicolor in the presence of organic peroxides, particularly dioxane peroxide, tetrahydrofuran peroxide and t-butylhydroperoxide, initiated free-radical copolymerization of acrylamide and lignin. Hydrogen peroxide showed no such effect. Both the type of peroxide and the catalytic efficiency of the enzyme were important to ensure a significant yield of copolymerisate and a high rate of acrylamide incorporation into a lignin backbone. The mechanism of the enzymatic grafting is discussed. Received: 12 August 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

Induction and functional role of cytochromes P450 in the filamentous fungi Mortierella alpina ATCC 8979 and Cunninghamella blakesleeana DSM 1906 during hydroxylation of cycloalkylbenzoxazoles

The occurrence and regulation of cytochrome P450 (P450) in Mortierella alpina and Cunninghamella blakesleeana have been studied to elucidate the enzymatic basis by which 2-cyclopentyl-1,3-benzoxazole is hydroxylated to 3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol by these organisms. The occurrence of P450 in M. alpina was first been shown after induction with n-hexane. An assay protocol was developed with n-hexane-induced cells and adapted to the handling of fungal mycelia. This allowed the direct spectral determination of P450 in non-fractionated whole-cell suspensions, and an investigation of its regulation. Small amounts of P450 have been detected in early-stationary-phase cells in the absence of exogenous inducers. Addition of 2-cyclopentyl-1,3-benzoxazole or n-hexane resulted in a significant induction of P450. Induction by n-hexane occurs in all phases of growth but decreases rapidly during the stationary phase. The rate of 2-cyclopentyl-1,3-benzoxazole hydroxylation correlated with the content of substrate-induced P450 but not with the level of n-hexane-induced P450. Hydroxylation rates were significantly diminished in the presence of typical P450 inhibitors, the interaction of which with P450 was shown with isolated microsomes of M. alpina. It is concluded that a P450 enzyme is responsible for the hydroxylation of 2-cyclopentyl-1,3-benzoxazole, but that multiple forms of P450 forms occur. Similarly, a dependence on P450 is shown by spectral as well as by inhibition studies for the hydroxylation of this substrate by C. blakesleeana. Received: 18 August 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

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     

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     

Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

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     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997