Production of indole-3-acetic acid by mutant strains of Ustilago maydis (maize smut / huitlacoche)

Production of indole-3-acetic acid (IAA) by four strains of the maize pathogen Ustilago maydis was analyzed. The fungus induces gall formation on its host plant and IAA production by  U. maydis may be required as a pathogenicity or virulence factor. The study included the FB2 wild-type strain and the 103, 130FZ and 130FT mutants. Results show that treatment with clofibric acid, alone or in combination with UV light, can be used to obtain  U. maydis strains with defective IAA production in vitro, as quantified with the Salkowski reagent and by HPLC. The strain with the lowest production was 130FT, and its peak IAA level represented only 16% of the highest value obtained for the FB2 wild-type strain (124 μg/ml). Received: 11 April 1996 / Received last revision: 5 September 1997 / Accepted: 11 September 1997     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

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     

Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli : A comparison of the synthesis capacities of L-form strains with an E. coli producer strain

The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Yields of 45–75 mg/l total antibody protein and of 10–18 mg/l functional miniAb were estimated in the growth medium of shaking cultures 40–80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound. The yields of functional miniAb could be optimized by lowering the growth temperature from 37 °C to 26–32 °C and by supplementation of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2–4 mg functional miniAb/g cell dry weight) to that of the producer strain E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without the periplasmic compartment. Received: 14 April 1997 / Received revision: 17 July 1997 / Accepted: 25 August 1997     

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

 In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l^–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l^–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l^–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l^–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex. Received: 2 February 2000 / Revision received: 30 March 2000 / Accepted: 22 June 2000     

A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium. Received: 29 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Limonene bioconversion to high concentrations of a single and stable product, perillic acid, by a solvent-resistant Pseudomonas putida strain

The white-rot basidiomycete Phanerochaete chrysosporium BKM-F-1767 was tested for its capacity to degrade dehydroabietic acid (DHA). In anaerobic treatment, this molecule is the most recalcitrant member of the resin acid group, which is known to cause operational problems to anaerobic reactors treating pulp and paper industry wastewaters. In this study the effect of DHA on different parameters, such as growth, ligninolytic enzyme activity, extracellular protein production as well as both glycerol and ammonium consumption by the fungus, was determined. Although the above parameters were affected by the addition of DHA, the results show that the fungus could still produce significant titres of ligninolytic enzymes. The fungus removed 47% of the DHA initially present in the static culture, after 10 days of incubation. Anaerobic toxicity assays showed that the treatment of DHA with P. chrysosporium reduced the methanogenesis and acetogenesis inhibition caused by DHA and allowed improved methane production by the anaerobic bacteria. Received: 10 June 1997 / Received revision: 6 January 1998 / Accepted: 24 January 1998     

Production of Spirulina sp. in sea water supplemented with anaerobic effluents in outdoor raceways under temperate climatic conditions

The use of untreated sea water supplemented with anaerobic effluents from digested pig waste and sodium bicarbonate was evaluated as a low-cost medium for semi-continuous cultivation of a mixed culture of two Spirulina strains in outdoor raceways under temperate climatic conditions (pond temperature in the range 21–26 °C and light intensity in the range 225–957␣μE m⁻² s⁻¹). The mixed culture had a predominant population (86.6 ± 3.9%) of an atypical Spirulina strain consisting of straight filaments, which appeared spontaneously after the strain with helicoidal trichomes had been subcultured. Morphological studies for the identification of the type and size of trichomes of the two strains (HF and SF) were carried out. The proportions of the two strains were observed to be stable during the monitoring period (30 days). Three different sets of semicontinuous cultures were carried out. Sets 1 and 2 were operated under regime 1 (a single addition of anaerobic effluents at time zero and no pH control) during the same season (June and July) of different years. Set 3 was operated under regime 2 (semi-continuous addition of anaerobic effluents and pH control) during the autumn. A minimum productivity of 3.6 g m⁻² day⁻¹ was obtained at one of the lowest temperatures (22.1 °C) and light intensities (245 μE m⁻² s⁻¹) and a maximum productivity of 10.9 g m⁻² day⁻¹ was observed at the highest temperature (25 °C) and highest average light intensity (618 μE m⁻² s⁻¹) registered for sets 1 and 2. The protein content in the Spirulina biomass harvested from these two sets varied from 17% to 65.6%. In set 3, a maximum productivity of 9.0 g m⁻² day⁻¹ was recorded at an average temperature of 24.4 °C and at an average light intensity of 668 μE m⁻² s⁻¹. The protein content in this set under regime 2 varied within a narrower range than in set 1 and set 2 (from 34.8% to 49.1%), apparently because of a continuous availability of ammonia nitrogen at a level of 30–50 mg l⁻¹. However, in terms of the removal of ammonia nitrogen and chemical oxygen demand, regime 1 was more efficient than regime␣2. Received: 3 September 1996 / Received revision: 19 February 1997 / Accepted: 7 March 1997     

Production of flavour compounds by yogurt starter cultures

The present work studied the production of carbonyl compounds and saturated volatile free fatty acids by pure cultures of Streptococcus thermophilus and Lactobacillus bulgaricus, and by starter cultures for Bulgarian yogurt during cultivation and cooling. The mixed cultures formed volatile aromatic compounds more actively than the pure cultures. A guiding factor in the preparation of the starter cultures was the biochemical activity of Lactobacillus bulgaricus in synthesizing the major carbonyl compounds, acetaldehyde, diacetyl and the volatile fatty acids C₂–C₁₀. The activity of the yogurt cultures in synthesizing carbonyl compounds was at its highest during milk coagulation and cooling, up to 7 h. However, maximum concentration was reached by 22–31 h. In the cooled 22–h starter cultures, acetaldehyde predominated (1415.0–1734.2 μg per 100 g) followed by diacetyl (165.0–202.0 μg per 100 g), acetoin (170.0–221.0 μg per 100 g), acetone (66.0–75.5 μg per 100 g), ethanol (58.0 μg per 100 g), and butanone-2 (3.6–3.8 μg per 100 g). The thermophilic streptococcus and lactobacillus cultures, and the starter cultures contained predominantly acetic, butyric and caproic acids. Received 19 June 1997/ Accepted in revised form 10 January 1998     

The amelioration of aluminium toxicity by silicon in wheat (Triticum aestivum L.): malate exudation as evidence for an in planta mechanism

Two wheat (Triticum aestivum L.) cultivars, one aluminium tolerant (Atlas 66) and one sensitive (Scout 66), were grown in a continuous-flow culture system (≤pH 5.0) containing aluminium (0–100 μM) and silicon (0–2000 μM) in factorial combination. Treatment with silicon resulted in a highly significant amelioration of aluminium toxicity as assessed by root growth in both cultivars. Amelioration was influenced by wheat cultivar and silicon concentration, as 2000 μM silicon significantly ameliorated the toxic effects of 100 μM aluminium in Atlas 66, and only 5 μM silicon alleviated the effect of 1.5 μM aluminium on Scout 66. Nutrient medium pH was critical, as an amelioration by silicon was apparent only at pH > 4.2 for Atlas 66, and at pH > 4.6 for Scout 66. Silicon neither reduced levels of toxic aluminium species in the growth solutions, nor the amount of aluminium taken up by roots. In experiments to assess exudation of malate by Atlas 66 roots treated with 100 μM aluminium, the presence of 2000 μM silicon (pH 4.6) was found to have a negligible effect on exudation. In contrast, citrate, a known aluminium chelator, reduced aluminium-induced exudation of malate at 5–40 μM and completely inhibited it at 100 μM citrate. The results indicate that silicon does not reduce aluminium phytotoxicity as a result of aluminium/silicon interactions in the external media, and that the mechanism of amelioration has an in planta component. Received: 22 April 1997 / Accepted: 16 August 1997     

Morphogenesis and regeneration from stomatal guard cell complexes of cotton (Gossypium hirsutum L.)

 The development of a regeneration system from cotton stomatal guard cells directly on epidermal strips is described. The most important factors affecting embryogenic callus initiation in both of the varieties tested (Coker 312 and 315) were the source of the epidermal tissue, including plant age (4–5 months old), the developmental stage of the flower (opening flower stage) from which bracts were obtained, the composition of the culture medium and light irradiance. The flower developmental stage was critical for callus formation, which was observed only from bracts obtained from opening flowers. In addition, epidermal strips excised from the bract basal region were more responsive in culture than those obtained from the top region. Improved callus initiation was obtained on epidermal strips which had their cuticle in contact with the culture medium. Light irradiance was a limiting factor for embryogenic callus formation, which was observed only in calluses cultured under the lower light irradiance (15.8 μmol m^–2 s^–1). Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing naphthalene acetic acid (10.7 μM) and isopentenyladenine (4.9 μM). Histodifferentiation of somatic embryos was improved on a medium containing naphthaleneacetic acid (8.1 μM)+isopentenyladenine (2.5 μM) and abscisic acid (0.19–0.38 μM). Somatic embryo germination and plantlet development were obtained using established protocols with few modifications. On average, one fully developed plant was obtained from the culture of circa 100 epidermal strips in both cultivars. Received: 19 May 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

On-line enzyme activity determination using the stopped-flow technique: application to laccase activity in pulp mill waste-water treatment

An automated system for on-line measurement of enzyme activity is proposed. The system uses a flow injection manifold in the stopped-flow mode to measure initial reaction rates. The time during which the flow is halted is selected in such a way as to optimise the enzyme/substrate ratio for the correct determination of activity values. The proposed system was used to determine the activity of laccase produced by the fungus Trametes versicolor immobilised on nylon in a fixed-bed reactor used for treating pulp mill waste water. Received: 17 February 1997 / Received revision: 23 April 1997 / Accepted: 27 April 1997     

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB⁻4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains. Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Biodegradation of the pesticide 4,6-dinitro-ortho-cresol by microorganisms in batch cultures and in fixed-bed column reactors

A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and 84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly ¹⁴C-labelled DNOC was recovered as ¹⁴CO₂. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol, 2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads in fixed-bed column reactors were 30 mmol day⁻¹ (l reactor volume)⁻¹, leaving an effluent concentration of less than 5 μg l⁻¹ DNOC in the outflowing medium. The apparent K _s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing mechanism. Received: 27 March 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997     

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     

Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D=0.05 h⁻¹) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive. Received: 24 February 1997 / Received revision: 22 May 1997 / Accepted: 25 May 1997