Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger

Summary Growth, citric acid production and enzymatic activity of the mitochondrial respiratory enzymes of a wild-type and a citric-acid-producing mutant of Aspergillus niger have been compared during fermentation under citric-acid-accumulating and non-accumulating conditions. Under non-accumulating conditions, both strains showed standard growth and no citric acid production. The mutant strain was characterized by delayed onset of growth and lowered cell yield. Under citric-acid-accumulating conditions the wild-type strain exhibited decelerated growth and a maximal citric acid concentration of 12 g l^–1. Reduced, but continuing growth and citric acid production of 32 g l^–1 was observed for the mutant strain. In general, the mutant strain exhibited reduced activity for the proton-pumping respiratory complexes and enhanced activity for the alternative respiratory enzymes. In contrast to the stable activity of complex I in the wild-type strain, this complex was selectively lost in the mutant strain at the onset of citric acid production, while the alternative NADH dehydrogenases were kept at enhanced and constant activity. A possible causal connection between the loss of complex I and citric acid accumulation is discussed. Offsprint requests to: J. Wallrath     

Production of acetate by mutant strains of Clostridium thermoaceticum

Summary Mutant strains were derived from Clostridium thermoaceticum ATCC 39 289 by treatment with chemical mutagenic agents and selective enrichment procedures. Some mutant strains exhibited growth when cultured in media containing 20 mabetm (1.75 g l^–1) pyruvate of high-magnesium lime (dolime) above pH 6.0. One strain (G-20) grew and produced acetate when 80 mabetm (7 gl^–1) pyruvate or 50 mabetm (2.3 g l^–1) formate at pH 5.6 was the sole energy source. In a fed-batch process controlled at pH 6.2, this mutant produced 52.5 g l^–1 acetate (equivalent to 72.5 g l^–1 Na acetate) and 67 g l^–1 calcium-magnesium acetate (CMA) in 140 h when dolime was the neutralizing agent, with 93% substrate utilization. This mutant strain holds promise for CMA production due to its better tolerance of dolime and its ability to synthesize high levels of acetic acid. Offprint requests to: S. R. Parekh     

A novel <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> mutant with high glycerol productivity in high phosphate concentration medium

A novel Candida glycerinogenes mutant, which possesses high glycerol productivity in a high phosphate concentration medium, was obtained by mutagenesis of an industrial glycerol producer. The mutant accumulated a total biomass of 11.5 g l⁻¹, which is less than the 15 g l⁻¹of the wild-type strain, but it consumed glucose faster than the wild-type strain did. The mutant reached its maximal glycerol concentration of 129 g l⁻¹ in 84 h compared to 96 h for the wild-type strain. High cytoplasmic glycerol-3-phosphate dehydrogenase activity of the mutant in the early glycerol formation phase, leading to a rapid glycerol synthesis and accumulation, may be the main reason for the short fermentation process.     

Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l^–1 h^–1). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l^–1 h^–1 of at 30 °C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

Isolation and study of two strains ofLeptospirillum-like bacteria from a natural mixed population cultured on a cobaltiferous pyrite substrate

Two strains ofLeptospirillum-like bacteria, L6 and L8, have been isolated from a mixed inoculum, also containingThiobacillus ferrooxidans andT. thiooxidans, cultured for one year with a colbaltiferous pyrite as energy substrate in a 100 I continuous bioleaching laboratory unit. Several physiological properties of the strains are described. The vibrio-shaped microorganisms grew at pH values lower than 1.3. Their growth rate was maximum between 2.5 and 8.0 g l¹ ferrous iron. The optimal growth temperature was 37.5° C. Ferric iron had a stimulative effect on bacterial development up to 8 g l^–1, and growth was as rapid at 14 g l^–1 ferric iron as at 8 g l^–1. The negative influence of cobalt on the final cell concentration was observed at 0.5 g l^–1, but the growth rate was not affected up to 2 g l^–1. The G + C content of strains L8 is 55.6 mol%.     

2-Propanol degradation by Sulfolobus solfataricus

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 °C with or without glucose at 10 g l^–1. The presence of 3.92 g 2-propanol l^–1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l^–1 increased 2-propanol and acetone utilization from 0.93 g l^–1 to 1.77 g l^–1 and from 0.11 g l^–1 to 1.62 g l^–1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l^–1 h^–1, compared to 0.0012 g l^–1 h^–1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

Production of cellulases in batch culture using a mutant strain of Trichoderma reesei growing on soluble carbon source

Trichoderma reesei Rut-C30 is a highly derepressed mutant which synthesised active cellulases in culture media containing glucose and lactose as the only carbon sources. The maximum biomass, filter paper and specific filter paper activities for cell growth on 20 g glucose l^–1 were 20 g dry cell wt l^–1, 1.9 FPU ml^–1 and 4.8 FPU mg^–1 protein respectively, while on 40 g glucose l^–1 were 25 g dry cell wt l^–1, 4.5 FPU ml^–1 and 6.2 FPU mg^–1 protein, respectively. This strain had a higher specific filter paper activity (6.2 FPU mg^–1 protein) than was produced by other T. reesei mutants (3.6 FPU mg^–1 protein).     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

Reducing by-product formation in L-lactic acid fermentation by Rhizopus oryzae

During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l^–1 to 0.6 g l^–1 KH₂PO₄ reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l^–1 to 71 g l^–1 and from 1.36 g l^–1 to 0.18 g l^–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l^–1 KH₂PO₄ was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production.     

Invertase production by Aspergillus niger in submerged and solid-state fermentation

Three Aspergillus nigerstrains were grown in submerged and solid state fermentation systems with sucrose at 100 g l^–1. Average measurements of all strains, liquid vs solid were: final biomass (g l^–1), 11 ± 0.3 vs 34 ± 5; maximal enzyme titres (U l^–1) 1180 ± 138 vs 3663 ± 732; enzyme productivity (U l^–1h^–1) 20 ± 2 vs 87 ± 33 and enzyme yields (U/gX) 128 ± 24 vs 138 ± 72. Hence, better productivity in solid-state was due to a better mould growth.     

Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations

Poly(3-hydroxybutyrate) (PHB) was produced by fed-batch cultures of Ralstonia eutropha with phosphate limitation under different glucose concentrations. When glucose was kept at 2.5 g l^–1, cell growth and PHB synthesis were limited due to the shortage of carbon source but a higher PHB content occurred in the cell-growth stage. This shows that a low glucose concentration is favorable for PHB accumulation in R. eutropha. PHB obtained with glucose at 9 g l^–1 is 1.6 times that obtained with 40 g l^–1. When glucose was in the range of 9 to 40 g l^–1, PHB concentration and productivity decreased significantly with the increase of glucose concentration. The highest PHB productivity was obtained with glucose at 9 g l^–1.     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium

Fourteen yeast strains were screened for production of 2-phenylethanol from l-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l^–1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l^–1 at 35 °C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.     

Mn2 + improves surfactin production by Bacillus subtilis

Surfactin productivity by Bacillus subtilis was increased from 0.33 g l^–1 to 2.6 g l^–1 by adding 0.01 mM Mn²⁺ to a defined glucose medium. The final yield exceeded that of most reported values for genetically improved strains.     

Oxalic acid production from lipids by a mutant of Aspergillus niger at different pH

Crude rapeseed oil and post-refining fatty acids were used as substrates for oxalic acid production by a mutant of Aspergillus niger. Both the final concentration and the yield of the product were highest at pH 4 to 5. With a medium containing 50 g lipids l^–1, production reached a maximum of 68 g oxalic acid l^–1 after 7 d. A high yield of the product (up to 1.4 g oxalic acid g^–1 lipids consumed) was achieved with oil and fatty acids combined.     

Oleic acid improves poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Ralstonia eutropha in inverted sugar and propionic acid

With the objective of verifying the influence of oleic acid as a nutritional supplement in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha, cultures were established with 0.3 g oleic acid l^–1 and without this supplement, in 30 g inverted sugar l^–1 and 1 g propionic acid l^–1. The use of this supplement increased the accumulation of polymer from 18.3% to 28.3% (w/w) although the mass of 3-hydroxyvalerate in the polymer remained constant for both cultures.     

Optimized production of L-β-hydroxybutyric acid by a mutant of Yarrowia lipolytica

A mutant strain of Yarrowia lipolytica was developed which produced 8.0 g l--hydroxybutyric acid l^–1 from butyric acid in a batch culture. The optimum culture conditions in the fermenter for maintenance of a high cell activity, determined by chemostat analyses, were a specific growth rate of 0.06 h^–1, a glucose concentration of 2.0 g l^–1, and a butyric acid concentration of 8.1 g l^–1. A fed-batch fermentation was performed under these conditions resulting in an l--hydroxybutyric acid yield of 31 g l^–1.