Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Antimicrobial activity of protoanemonin,a lactone from ranunculaceous plants

Protoanemonin, a component of Ranunculus bulbosus, was tested as an antifungal agent on selected strains of dermatophytes and yeasts. The minimum inhibitory concentrations ranged from 2.0 to 7.5×10^–4 M and the minimum lethal concentrations from 3.8×10^–4 M to >1.0×10^–3 M. The most sensitive dermatophyte tested was Epidermophyton floccosum, and the most sensitive yeast Rhodotorula glutinis. The effects of different culture media and of light on the sensitivity of Rhodotorula glutinis to protoanemonin were also tested. Structural analogies between protoanemonin and other cytotoxic unsaturated lactones, and the reversal by the amino acid cysteine of the antifungal action suggest a possible mechanism of action.     

Enhanced nisin and pediocin production on whey supplemented with different nitrogen sources

Production of nisin and pediocin were followed, respectively, in Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 grown with lactose and four different nitrogen sources. Neither NH₄Cl nor glycine improved production of the bacteriocins. Both yeast extract and Casitone increased pediocin production from 55 BU ml^–1 to 195 BU ml^–1 and 185 BU ml^–1, respectively. Nisin increased from 21 BU ml^–1 to 74 BU ml^–1 and 59 BU ml^–1 with these nitrogen sources.     

Xylitol production from aspenwood hemicellulose hydrolysate by Candida guilliermondii

The production of xylitol by the yeast Candida guilliermondii was investigated in batch fermentations with aspenwood hemicellulose hydrolysate and compared with results obtained in semi-defined media with a mixture of glucose and xylose. The hemicellulose hydrolysate had to be supplemented by yeast extract and the maximum xylitol yield (0.8 g g^–1) and productivity (0.6 g l^–1 h^–1) were reached by controlling oxygen input.     

Efficacy of seed-based media for the mould-yeast conversion of Blastomyces dermatitidis

The efficacy of 20 seed-based media is reported for the in vitro mould-yeast conversion of Blastomyces dermatitidis, employing pharmamedia agar, peptone glucose agar, glucose agar and water agar as controls. The mould-yeast conversion varied significantly according to the culture medium, fungal strains and incubation period (p<0.01). Garden-pea, chick-pea, cow-pea, soyabean, peanut, green gram, French bean, lentil, okra and cottonseed converted all of the 7 B. dermatitidis test strains after 5 days of incubation at 37 ° C. Although the efficacy of many of these seed media was found to be at par with pharmamedia agar — a commercial cottonseed embryo-derived protein, garden-pea seed agar is adopted because of the wider availability and low fat content of this seed. The recommended composition of the medium comprises 2% aqueous seed extract, 2% glucose and pH 6–7. Only nigerseed and sunflower seeds failed to support the conversion of B. dermatitidis. Of the control media, peptone glucose agar, glucose agar and water agar did not support the conversion of 2 of the B. dermatitidis test strains. The mechanism underlying variable mould-yeast conversion of B. dermatitidis on seed-based media is not clearly understood. However, most of the seeds supporting excellent mould-yeast conversion are known for their high protein content. The conversion was apparently not affected by the fat content of the seeds or by incorporation of glucose in the medium.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Population growth kinetics of the nematode, Steinernema feltiae, in submerged monoxenic culture

Monoxenic cultures of the nematode, Steinernema feltiae, were carried out on two complex liquid media: P1, mainly soybean flour/egg yolk/yeast extract, and P2, mainly egg yolk/yeast extract. Up to 140 000–200 000 nematodes ml^–1 were produced within 7 days, and more than 95% of the final population was in the infective juvenile stage. The total nematode concentration growth curve had a sigmoidal shape. Nematode population growth kinetics were modelled using a re-parameterised Gompertz model. Yeast extract concentration appeared to be a key factor for obtaining high nematode concentrations.     

Growth promotion of Prunus rootstocks by root treatment with specific bacterial strains

A collection of bacterial strains obtained from a wide-range origin was screened for ability to promote growth in two types of Prunus rootstocks in a commercial nursery. Only few strains promoted growth significantly and consistently, and a strong specificity for the rootstock cultivar was observed. Irrigation of plants with Pseudomonas fluorescens EPS282 and Pantoea agglomerans EPS427 significantly increased plant height and root weight of the plum Marianna 2624 and the peach–almond hybrid GF-677, respectively. Plant height showed a higher rate of growth in early stages of development (2.6–3.5 times the non-treated controls), but the effect decreased with plant age. However, in aged plants growth promotion was more significant on root weight (1.9 times the non-treated controls) than on plant height. The efficacy of growth promotion and the persistence of strains in the root environment were dependent on the bacterial inoculum concentration applied. Increases in root development were maximum at inoculum concentrations of up to 8 log₁₀ CFU ml^–1 (ca 10 log₁₀ CFU L^–1 of potting mix). Population levels at the optimum inoculum concentration were around 7 log₁₀ CFU g f.w.^–1 root material at early stages of development and decreased to 4 log₁₀ CFU g f.w.^–1 after several months of development. The best plant growth-promoting strains were very diverse in secondary metabolite production and antagonistic ability against several plant pathogens.     

Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

Degradation of polyaromatic hydrocarbons by Burkholderia cepacia 2A-12

A new strain of bacterium degrading polyaromatic hydrocarbons (PAHs), Burkholderia cepacia 2A-12, was isolated from oil-contaminated soil. Of three PAHs, the isolated strain could utilize naphthalene (Nap) and phenanthrene (Phe) as a sole carbon source but not pyrene (Pyr). However, the strain could degrade Pyr when a cosubstrate such as yeast extract (YE) was supplemented. The PAH degradation rate of the strain was enhanced by the addition of other organic materials such as YE, peptone, glucose, and sucrose. YE was a particularly effective additive in stimulating cell growth as well as PAH degradation. When 1 g YE l^–1, an optimum concentration, was supplemented into the basal salt medium (BSM) with 215 mg Phe l^–1, the specific growth rate (0.30 h^–1) and Phe-degrading rate (29.6 mol l^–1 h^–1) were enhanced approximately ten and three times more than those obtained in the BSM with 215 mg Phe l^–1, respectively. Both cell growth and PAH degradation rates were increased with increasing Phe and Pyr concentrations, and B. cepacia 2A-12 had a tolerance against Phe and Pyr toxicity at the high concentration of 730–760 mg l^–1. Through kinetic analysis, the maximum specific growth rate ( _max) and PAH degrading rate ( _max) for Phe were obtained as 0.39 h^–1 and 300 mol l^–1 h^–1, respectively. Also, _max and _max for Pyr were 0.27 h^–1 and 52 mol l^–1 h^–1, respectively. B. cepacia 2A-12 could simultaneously degrade crude oil as well as PAHs, indicating that this bacterium is very useful for the removal of oils and PAHs contaminants.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Brachionus calyciflorus Pallas as agent for the removal of E. coli in sewage ponds

Brachionus calyciflorus (Pallas) is a common brachionid in sewage oxidation ponds. The uptake and assimilation of E. coli was optimal at concentrations of 2.7–6.9 × 10⁸ cells ml^–1 while assimilation coefficient per body weight of B. calyciflorus was found to be 10% · Ind.^–1 d^–1. More than two eggs per individual were produced during 24 hours when brachionids were fed with a mixutre of E. coli (10⁹ cells · ml^–1) and Chlorella spp. (10⁶ cells · ml^–1). The nutritional value of the mixture of E. coli and Chlorella spp. was found to be higher than that of bacteria alone.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4--d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca²⁺, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4--d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h^–1, biomass yield coefficient of 0.5 g g^–1 and feed substrate concentration of 200 g L^–1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L^–1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g^–1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.Nomenclature C _ox Dissolved oxygen concentration (mg L^–1) - CPR Carbon dioxide production rate (mmol h^–1) - C _s0 Glucose concentration in the feed (g L^–1) - C _s Substrate concentration in the fermenter (g L^–1) - C _s.crit Critical substrate concentration (g L^–1) - E Ethanol concentration (g L^–1) - F _s Substrate flow rate (g h^–1) - i Sample number (–) - K _e Constant in Equation 6 (g L^–1) - K _o Constant in Equation 7 (mg L^–1) - K _s Constant in Equation 5 (g L^–1) - m Specific maintenance term (h^–1) - OUR Oxygen up-take rate (mmol h^–1) - q _ox Specific oxygen up-take rate (h^–1) - q _ox.max Maximum specific oxygen up-take rate (h^–1) - q _p Specific product formation rate (h^–1) - q _s Specific substrate up-take rate (g g^–1 h^–1) - q _s.max Maximum specific substrate up-take rate (g g^–1 h^–1) - RQ Respiratory quotient (–) - S Total substrate in the fermenter at timet (g) - S ₀ Substrate mass fraction in the feed (g g^–1) - t Fermentation time (h) - V Instantaneous volume of the broth in the fermenter (L) - V ₀ Starting volume in the fermenter (L) - V _si Volume of samplei (L) - x Biomass concentration in the fermenter (g L^–1) - X ₀ Total amount of initial biomass (g) - X _t Total amount of biomass at timet (g) - Y _p/s Product yield coefficient on substrate (–) - Y _x/e Biomass yield coefficient on ethanol (–) - Y _x/s Biomass yield coefficient on substrate (–) Greek letters Moles of carbon per mole of yeast (–) - Moles of hydrogen atom per mole of yeast (–) - Moles of oxygen atom per mole of yeast (–) - Moles of nitrogen atom per mole of yeast (–) - Specific growth rate (h^–1) - _crit Critical specific growth rate (h^–1) - _E Specific ethanol up-take rate (h^–1) - _max.E Maximum specific ethanol up-take rate (h^–1)     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible     

Endogenous ethylene and reversing methyl jasmonate inhibition of Amaranthus caudatus seed germination by benzyladenine or gibberellin

BA at 10^–5 M, GA₃ at 3×10^–4 M or GA₄₊₇ at 3×10^–5 M partially or largely reversed the inhibition of Amaranthus caudatus seed germination due to JA-Me. BA or GA₃ did not affect ethylene production and ACC oxidase activity in vivo in the presence of JA-Me before radicle protrusion. However, both increased ethylene production after 72 h of incubation, when the reversal of the JA-Me inhibition of seed germination was observed. AVG at 3×10^–4 M decreased ethylene production when it was applied simultaneously with BA and JA-Me or GA₃ and JA-Me, but it had no effect on seed germination. NBD almost completely reversed the stimulatory effect of BA, GA₃ or GA₄₊₇ on the germination of seeds in the presence of JA-Me. Exogenous ethylene reversed the inhibitory effect of NBD. The results indicate that action of endogenous ethylene is involved in the response of JA-Me inhibited seeds to BA or GA_s.     

Control of xylanase production without protease activity in Bacillus sp. by selection of nitrogen source

Maximum xylanase activity, of 380 IU ml^–1, with negligible protease activity, occurred when Bacillus SSP-34 was grown for 96 h with yeast extract and peptone each at 0.25%. Other concentrations of the combination gave xylanase activities less than 66% of that with the optimum nitrogen source concentration and protease activities in the range of 0.01–0.045 IU ml^–1.