Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media

Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch was necessary, indicating high activity of the clostridial amylases produced by the strain applied. Received: 17 April 1998 / Received revision: 22 June 1998 / Accepted: 27 June 1998     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

Process development for high-level secretory production of carboxypeptidase Y by Saccharomyces cerevisiae

In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level, resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH 6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously reported. The process developed could be easily scaled-up to industrial-scale fermentation. Received: 16 January 1998 / Received revision: 16 February 1998 / Accepted: 27 February 1998     

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

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     

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     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Effect of enclosing rocks and aeration on methanogenesis from coals

Two coals of different rank, mined in Russia, were treated by an anaerobic methanogenic enrichment culture. The addition of alkaline enclosing rock to the lower-rank coal increased the pH of the incubation medium and methane production above that of the higher-rank coal with addition of its enclosing rock. This effect was accompanied by the leaching of cations from the incubation medium. The coal was processed without a preliminary chemical treatment in a two-stage aerobic/anaerobic bioreactor containing an anaerobic methanogenic granulated enrichment culture. Received: 15 January 1998 / Received revision: 2 October 1998 / Accepted: 2 October 1998     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

An enzyme-linked immunosorbent assay for the estimation of fungal biomass during solid-state fermentation

An enzyme-linked immunosorbent assay for sensitive, specific and quantitative estimation of fungal biomass during solid-state fermentation is described. Using this method, differential growth rates and colonization of the substrate can be studied. The assay has potential application for the efficient monitoring of solid-state fermentation involving specific fungus, for which available methods are not adequate. Received: 18 November 1997 / Received last revision: 8 June 1998 / Accepted: 14 June 1998     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Utilisation of saccharides in extruded domestic organic waste by Clostridium acetobutylicum ATCC 824 for production of acetone, butanol and ethanol

Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production. Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively. The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824. This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement. This strain produced 4 g ABE/100 g extruded DOW. When C. acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and β-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW. The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria. These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium. Received: 5 November 1999 / Received revision: 21 February 2000 / Accepted: 25 February 2000     

A genetically engineered strain of Saccharopolyspora erythraea that produces 6,12-dideoxyerythromycin A as the major fermentation product

The erythromycin producer, Saccharopolyspora erythraea ER720, was genetically engineered to produce 6,12-dideoxyerythromycin A, a novel erythromycin derivative, as the major macrolide in the fermentation broth. Inspection of the biosynthetic pathway for erythromycin would suggest that production of this compound could be achieved simply through the disruption of two genes, that encoding the erythromycin C-6 hydroxylase (eryF ) and that encoding the erythromycin C-12 hydroxylase (eryK ). The double mutant, however, was found to produce a mixture of 6,12-dideoxyerythromycin A and the precursor, 6-deoxyerythromycin D. Complete conversion to the desired product (to the limit of detection by TLC) was achieved by inserting an additional copy of the eryG gene, encoding the erythromycin 3′′-O-methyltransferase and driven by the ermE* promoter, into the S. erythraea chromosome. Received: 6 October 1997 / Received revision: 27 January 1998 / Accepted: 24 February 1998     

Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropropane, and varied from 0.07 to 1.03 ml min⁻¹ mg of cells⁻¹ for 1,2,3-trichloropropane and 1-chloropropane respectively. Turnover-dependent inactivation occurred for all of the chloropropanes tested. The inactivation constants were lower for 1-chloropropane and 1,2-dichloropropane than for 1,2,3-trichloropropane and 1,3-dichloropropane. Not all the chloride was released during cometabolic transformation of the chlorinated propanes and production of monochlorinated- and dichlorinated propanols was found by gas chromatography. The reaction pathway of 1,2,3-trichloropropane conversion was studied by mass spectrometric analysis of products formed in ²H₂O, which indicated that 1,2,3-trichloropropane was initially oxidized to 2,3-dichloropropionaldehyde and 1,3-dichloroacetone, depending on whether oxygen insertion occurred on the C-3 or C-2 carbon of 1,2,3,-trichloropropane, followed by reduction to the corresponding propanols. The results show that chloropropanes are susceptible to cometabolic oxidation by methanotrophs, but that the transformation kinetics is worse than with cometabolic conversion of trichloroethylene. Received: 27 November 1997 / Received revision: 27 February 1998 / Accepted: 27 February 1998     

Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis

Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions. This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space. Received: 27 February 1998 / Received revision: 21 August 1998 / Accepted: 3 September 1998     

A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation

We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisherilux operon. This recA–lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment. Received: 18 February 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998     

Bioconversion of 2-cyanopyrazine to 5-hydroxypyrazine-2-carboxylic acid with Agrobacterium sp. DSM 6336

5-Hydroxypyrazine-2-carboxylic acid, a versatile building block for the synthesis of new antituberculous agents, was prepared by whole-cell biotransformation from 2-cyanopyrazine via pyrazinecarboxylic acid using Agrobacterium sp. DSM 6336. By developing a fermentation process for this two-enzyme-step bioconversion, a product concentration of 286 mM (40 g/l) was obtained. After the isolation method had been optimized the total yield was 80%. Received: 28 February 1997 / Received revision: 28 April 1997 / Accepted: 4 May 1997     

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