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     

Inactivation of bacteriophage k, Escherichia coli, and Candida albicans by ozone

The effects of ozone (O₃) on three types of microbes were studied. Test suspensions were exposed to 600 ppm O₃ at room temperature. Control experiments were performed under identical conditions using oxygen gas. Bacteriophage λ was completely inactivated at 10 min while Escherichia coli and Candida albicans were only inactivated by factors of 10⁵ and 10⁴ respectively at 40 min. Exposure of a mixed microbial suspension to O₃ for 5 min resulted in 100% killing of bacteriophages while the viability of E. coli remained unchanged. Various body fluids containing phages were exposed to O₃. Compared to buffered solution, the decrease in phage titers was significantly slower in whole blood, plasma, and albumin. Both E. coli and  C. albicans had increased production of thiobarbituric-acid-reactive substances with increased O₃ exposure. ³H-labelled amino acids were incorporated into E. coli. O₃ treatment resulted in a loss of radioactivity, indicating leakage of cytoplasmic contents. The data indicate that microbes are inactivated by O₃ at different rates, possibly related to differential membrane permeability. The milieu in which microbes are present determines the effectiveness and outcome of O₃ treatment. Received: 15 October 1997 / Accepted: 24 February 1998     

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI ^q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide). Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996     

Structure and O2 uptake properties of a novel nickel(II) complex of pyridyl-pendant dioxocyclam [1-(2-pyridyl)methyl-5,7-dioxo-1,4,8,11-tetraazacyclotetradecane]

 Novel potentially five-coordinate pyridyl–pendant dioxocyclam [1-(2-pyridyl)methyl-5,7-dioxo-1,4,8,11-tetraazacyclotetradecane (H₂L) and its homologs (6-methyl and 6,6-dimethyl derivatives)] have been synthesized to study nickel(II) complexation. A purple nickel(II) complex with a deprotonated amide (NiHL) was isolated from aqueous equimolar solution of H₂L and Ni(ClO₄)₂. A yellow nickel(II) complex with two deprotonated amides (NiL) was crystallized from an H₂O/CH₃CN solution of H₂L and Ni(OH)₂. The X-ray crystal study of NiL showed a square-planar nickel(II) complex with the pyridyl–pendant remaining uncoordinated. It is concluded from the visible absorption and NMR study of NiL in aqueous solution that the four-coordinate NiL is in equilibrium with a five-coordinate square-pyramidal nickel(II) complex with the apical coordination of the pyridyl–pendant. A voltammetric study disclosed a low nickel(II/III) redox potential of +0.29 V vs SCE for NiL at pH 9.5 and 25  °C with 0.10 M Na₂SO₄. The nickel(II) complex NiL absorbed an equimolar amount of O₂ at pH 9.5 and 25  °C, and the O₂ was activated to cleave plasmid DNA. Received: 5 August 1996 / Accepted: 24 October 1996     

Bacterial magnetosomes: microbiology, biomineralization and biotechnological applications

Magnetotactic bacteria orient and migrate along geomagnetic field lines. This ability is based on intracellular magnetic structures, the magnetosomes, which comprise nanometer-sized, membrane-bound crystals of the magnetic iron minerals magnetite (Fe₃O₄) or greigite (Fe₃S₄). Magnetosome formation is achieved by a mineralization process with biological control over the accumulation of iron and the deposition of the mineral particle with specific size and orientation within a membrane vesicle at specific locations in the cell. This review focuses on the current knowledge about magnetotactic bacteria and will outline aspects of the physiology and molecular biology of the biomineralization process. Potential biotechnological applications of magnetotactic bacteria and their magnetosomes as well as perspectives for further research are discussed. Received: 2 December 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999     

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5′-phosphate decarboxylase (URA3) or orotidine-5′-pyrophosphatase (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A␣transformation frequency of approximately 10⁴/μg DNA was obtained, which is tenfold higher than results described in earlier reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid. Received: 19 August 1996 / Received last revision: 30 April 1997 / Accepted: 4 May 1997     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19

The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H₂O₂ required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable. Received: 16 September 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Nitrate removal from drinking water using a membrane-fixed biofilm reactor

Biological treatment of drinking water is a cost-effective alternative to conventional physico/chemical processes. A new concept was tested to overcome the main disadvantage of biological denitrification, the intensive post-treatment process to remove microorganisms and remnant carbon source. The biological reaction zone and carbon supply were separated from the raw water stream by a nitrate-permeable membrane. Denitrification takes place in a biofilm, which is immobilized at the membrane. In a series of bench-scale runs, different types of membranes and reactor configurations were investigated. The best denitrification rates achieved were 1230 mg NO₃ ⁻-N m⁻² day⁻¹. In one run, raw water containing 100 mg NO₃ ⁻ l⁻¹ was completely freed from nitrate. The membrane and the attached biofilm also represent a barrier against the passage of the C source and nutrients into the raw water. At concentrations of 20 mg l⁻¹ ethanol and 15 mg l⁻¹ phosphate in the bioreactor no diffusion through the membrane into the treated water was observed. Without any post-treatment, the effluent met nearly all the relevant criteria for drinking water; only the colony count was slightly increased. Received: 18 December 1996 / Received last revision: 14 April 1997 / Accepted: 19 April 1997     

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     

Development of a low-cost fermentation medium for ethanol production from biomass

Nutrient cost is an important aspect in the fermentation of biomass to ethanol. With a goal of developing a cost-effective fermentation medium, several industrially available nutrient sources were evaluated for their effectiveness in the simultaneous saccharification and fermentation of pretreated poplar with Saccharomyces cerevisiae D₅A. These studies showed that a low-cost medium containing 0.3% corn steep liquor and 2.5 mM MgSO₄ · 7H₂O was similar in performance to a nutrient-rich medium. Besides its low cost, this alternative medium consists of components that are available on a commercial scale, thereby making it industrially relevant. Received: 14 August 1996 / Received revision: 7 January 1997 / Accepted: 24 January 1997     

In vivo expression of a full-length cDNA copy of potato leafroll virus (PLRV) in protoplasts and transgenic plants

A full-length cDNA copy (PLRV_fl) of potato leafroll virus (PLRV) was constructed and examined in vivo for its biological activities by transient expression experiments with plasmid DNA or in vitro transcribed RNA. In addition, PLRV_fl cDNA was stably introduced into the genome of potato plants by Agrobacterium-mediated leaf disc transformation. Both transient and stable expression of PLRV_fl resulted in the synthesis of genomic and subgenomic PLRV RNAs. Transgenic plants accumulated the 17-kDa movement protein and displayed the typical symptoms of PLRV infection. This is the first example of the constitutive expression of a phloem-limited virus in planta. Received: 20 August 1996 / Accepted: 24 September 1996     

The action of antibiotics on the anaerobic digestion process

Antibiotics can disturb the production of biogas during anaerobic digestion. This study shows a systematic approach to understanding how the different bacterial populations involved in the final conversion of organic matter into methane are inhibited by 15 antimicrobial agents with different specificities and modes of action. The results obtained show the following trends: (i) some inhibitors, such as the macrolide erythromycin, lack any inhibitory effect on biogas production; (ii) some antibiotics, with different specificities, have partial inhibitory effects on anaerobic digestion and decrease methane production by interfering with the activity of propionic-acid- and butyric-acid-degrading bacteria,␣(e.g. antibiotics that interfere with cell wall synthesis, RNA polymerase activity and protein synthesis, especially the aminoglycosides); (iii) the protein synthesis inhibitors chlortetracycline (IC₅₀ 40 mg l⁻¹) and chloramphenicol (IC₅₀ 15–20 mg l⁻¹) are very powerful inhibitors of anaerobic digestion. The majority of the antibiotics tested lacked activity against acetoclastic methanogens, being active only on the acetogenic bacteria. However, chloramphenicol and chlortetracycline could cause the complete inhibition of the acetoclastic methanogenic archaea. Received: 6 February 1996 / Received revision: 24 July 1996 / Accepted: 5 August 1996     

Immobilisation of whole bacterial cells for anaerobic biotransformations

Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction. A flow-rate/activity plot was constructed for each flow-through reactor and RA_1/2 values (residence time corresponding to 50 % nitrite removal) calculated for each reactor type. Cells immobilised in flat and hollow-fibre membranes were the most effective (RA_1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50 % nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively. Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors. Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA_1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively). Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA_1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively). Received: 12 August 1996 / Received revision: 14 November 1996 / Accepted: 15 November 1996     

Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans

A gene for a synthetic protein-based polymer, G-(VPGVG)₁₁₉-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study represents the first attempt to express a synthetic gene (with no natural analog) in a fungus. Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

Controlled secretion into the culture medium of a hybrid -glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5 -derived transposon

A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColE1 plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10⁻⁵–10⁻⁶). By insertion of a Bacillus hybrid β-glucanase (bgl ) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. β-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extracellular production of β-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur. Received: 17 October 1996 / Received revision: 23 December 1996 / Accepted: 4 January 1997     

Application of the Plackett-Burman experimental design to evaluate nutritional requirements for the production of Colletotrichum coccodes spores

Colletotrichum coccodes is being examined as a biological weed control agent for velvetleaf (Abutilon theophrasti). A modified Richard's solution containing V-8 juice has been used to produce C. coccodes spores for growth-chamber and field experiments. Although C. coccodes sporulates well in this medium, V-8 is not available as a bulk commodity and is too expensive for commercial production. Eight substrates were evaluated as replacements for V-8 juice in modified Richard's solution. Soy protein and casamino acids were equal to V-8 juice for sporulation of C. coccodes. The Plackett-Burman experimental design was used to test the relative importance of various components of a complex medium based on soy protein on mycelium biomass production and sporulation of C. coccodes. A new medium composed of sucrose (20 g/l), soy protein (5 g/l), KNO₃ (5 g/l), KH₂PO₄ (5 g/l), MgSO₄ (2 g/l), CaCl₂ (0.5 g/l), and CuSO₄ (0.05 g/l) was selected as the base medium for further study in the development of a low-cost and effective medium for C. coccodes spore production. Received: 29 July 1996 / Received revision: 21 October 1996 / Accepted: 10 November 1996     

Manganese uptake and toxicity in magnesium-supplemented and unsupplemented Saccharomyces cerevisiae

The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing␣ stages of batch growth and during growth in the presence of differing magnesium concentrations. Excess Mg was primarily sequestered in vacuoles. Mn²⁺-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn²⁺ accumulation. For example, after 6 h incubation in the presence of 50 μM Mn²⁺, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium. These differences were further accentuated at higher Mn²⁺ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn²⁺ binding. Cellular Mg status also influenced Mn toxicity towards S. cerevisiae. During exposure to 5 mM Mn²⁺, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (10⁹ cells)⁻¹ occurred after approximately 1.6 h and 3.6 h respectively. In cells containing 3300 nmol Mg (10⁹ cells)⁻¹, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn²⁺. It is concluded that Mn²⁺ uptake and toxicity in S. cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity. Received: 15 May 1996 / Received revision: 13 September 1996 / Accepted: 22 September 1996     

Cell-dislodging methods under serum-free conditions

In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml⁻¹), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity. Received: 23 September 1996 / Received revision: 27 December 1996 / Accepted: 30 December 1996     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 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