Extracellular pH affects regulation of the pcbAB gene in Penicillium chrysogenum

The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture. Received: 10 May 1996 / Received revision: 14 October 1996 / Accepted: 25 October 1996     

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     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Phycocyanin,a new elicitor for capsaicin and anthocyanin accumulation in plant cell cultures

  Elicitors of both fungal and bacterial origin that is, polysaccharides, proteins and fatty acids, are widely used for enhancement of secondary metabolites in plant cell cultures. In the present study, phycocyanin – a natural blue pigment that is the major light-harvesting biliprotein in the blue-green alga Spirulina platensis– was used as an elicitor to enhance the accumulation of capsaicin and anthocyanin in Capsicum frutescens and Daucus carota cell cultures respectively. Phycocyanin at 0.3, 0.6 and 1.2 mg% in capsicum cell cultures elicited a more than two-fold increase in capsaicin content with maximum productivity of 192 μg/g fresh weight. Similarly in Daucus carota cell cultures a two-fold increase in anthocyanin content was obtained at 0.3 mg% with a maximum productivity of 24.8 mg% on a dry-weight basis. In both the systems, phycocyanin showed an early elicitation of secondary metabolites. Received: 15 December 1995 / Received last revision: 15 July 1996 / Accepted: 18 July 1996     

Fermentability of hemicelluloses extracted from municipal waste and commercial xylans by Clostridium sp.

The fermentability of commercial xylans and municipal waste hemicelluloses in the presence of Clostridium sp. (C.SAIV; ATCC 700188) has been evaluated. Teak, deal wood, banana stalk and bagasse of the municipal waste contained significant amounts (approx. 12 %–23 %) of hemicellulose. Under optimized growth conditions, the growth rate of C.SAIV was improved as indicated by an increase in the concentration of ethanol in the culture broth. Commercial xylans were utilized fairly efficiently and ethanol formed from larch wood xylan and bagasse hemicellulose was at least 64 mM. The amount of ethanol formed from the bagasse hemicellulose was at least three times higher than any other reported value. The current study also indicated that the source and composition of hemicellulose played an important role in determining the fermentability of the substrate for some microorganisms. Received: 19 June 1996 / Received revision: 22 October 1996 / Accepted: 25 October 1996     

Aptitude of cheese bacteria for volatile S-methyl thioester synthesis I. Effect of substrates and pH on their formation by Brevibacterium linens GC171

The aptitude of resting cells of Brevibacterium linens G171 to synthesize S-methyl thioesters was studied in presence of methanethiol and nine short-chain fatty acids individually or as a mixture. Esterification of acetic, propionic and methyl branched-chain acids occurred with methanethiol alone and was enhanced by fatty acid addition. Addition of n-chain, 3-hydroxybutyric and 2-hydroxyvaleric acids allowed synthesis of n-chain thioesters up to thiocaproate. The kinetics of production and the effect of concentrations of both substrates and of cells were tested. The optimum pH for synthesis varied according to the kind of thioesters produced. Results suggested that thioesters were derived mainly from acyl-CoA from different metabolic breakdowns, such as the degradation of fatty acids or some amino acids, and that several acyltransferases could be involved. Received: 1 August 1996 / Received revision: 10 October 1996 / Accepted: 18 October 1996     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

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     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997     

Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp.

The marine photosynthetic bacterium Chromatium sp. successfully removed orthophosphate when grown phototrophically. The phosphate-uptake rate was almost constant at more than 5.0 mg- PO₄ ³⁻/l in synthetic medium. Addition of seawater causes flocculation of this strain. The successful use of seawater as an inexpensive source of magnesium could prove to be effective in the removal of photosynthetic bacterial cells from a medium. A semicontinuous culture system was used for the removal of low concentrations of phosphate and the phosphate-uptake activity of Chromatium sp. was maintained under 0.1 day⁻¹ dilution rate. This strain was also able to remove high concentrations of phosphate from domestic sewage. Received 24 May 1996 / Received revision: 5 August 1996 / Accepted: 6 September 1996     

Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6

Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni²⁺ and, to some extent, Zn²⁺ and Co²⁺, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities. Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

Influence of microbial concentration on the rheology of non-Newtonian fermentation broths

The objective of this study was to quantify the effect of fungal biomass concentration on the rheology of non-Newtonian fermentation systems. Batch fermentations of Penicillium chrysogenum were carried out with glucose as the sole carbon source. The flow behavior of the system was characterized at various fermentation times and was adequately described by the power-law model. The apparent viscosity of the fermentation broth was significantly affected by biomass concentrations in the fermenter. Fermentation broths containing 17.71 g/l biomass as dry weight were characterized by an apparent viscosity of 0.25 Pa s at a shear rate of 50 s⁻¹. Microbial concentration also affected the power-law flow-behavior index and the consistency index. The value of the consistency index ranged from 0.002 Pa s ⁿ at a biomass concentration of 0.1 g/l to 6.14 Pa s ⁿ at a biomass concentration of 17.71 g/l. The flow-behavior index decreased from an initial value of 1 to a final value of 0.17. Simple empirical correlations have been proposed to quantify the dependence of the power-law parameters on fungal biomass concentration. Experimental data obtained in this study were accurately described by these correlations. The general applicability of these relationships was tested, using previously published rheological data on Aspergillus awamori and Aspergillus niger fermentation broths, and good agreement was seen between experimental data and the predictions from the empirical correlations. Received: 24 March 1998 / Received revision: 10 September 1998 / Accepted: 16 October 1998     

Two-step degradation of pyrene by white-rot fungi and soil microorganisms

  The effect of soil microorganisms on mineralization of ¹⁴C-labelled pyrene by white-rot fungi in solid-state fermentation was investigated. Two strains of white-rot fungi, Dichomitus squalens and a Pleurotus sp., were tested. The fungi were incubated on milled wheat straw contaminated with [¹⁴C]pyrene for 15 weeks. CO₂ and ¹⁴CO₂ liberated from the cultures were determined weekly. To study the effect of soil microorganisms on respiration and [¹⁴C]pyrene mineralization in different periods of fungal development, the fungal substrate was covered with soil at different times of incubation (after 0, 1, 3, 5, 7, 9 or 11 weeks). The two fungi showed contrasting ecological behaviour in competition with the soil microflora. Pleurotus sp. was highly resistant to microbial attack and had the ability to penetrate the soil. D. squalens was less competitive and did not colonize the soil. The resistance of the fungus was dependent on the duration of fungal preincubation. Mineralization of [¹⁴C]pyrene by mixed cultures of D. squalens and soil microorganisms was higher than by the fungus or the soil microflora alone when soil was added after 3 weeks of incubation or later. With Pleurotus sp., the mineralization of [¹⁴C]pyrene was enhanced by the soil microflora irrespective of the time of soil application. With D. squalens, which in pure culture mineralized less [¹⁴C]pyrene than did Pleurotus sp., the increase of [¹⁴C]pyrene mineralization caused by soil application was higher than with Pleurotus sp. Received: 8 March 1996 / Received revision: 1 July 1996 / Accepted: 8 July 1996     

Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli

Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies. Received: 31 May 1996 / Received revision: 11 September 1996 / Accepted: 13 October 1996     

Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222

Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO₃ with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days. Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551

We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning. Received: 28 October 1996 / Received revision: 29 December 1996 / Accepted: 4 January 1997