Growth requirements and the establishment of a chemically defined minimal medium inZymomonas mobilis

Summary A chemically defined minimal medium which fulfils the growth requirements of differentZymomonas mobilis strains has been established. The kinetics of ethanol production of the strains ATCC 10988, CU1, CP4 and 11163 grown on the minimal medium at different glucose concentrations were measured. All strains produced ethanol at rates similar to those on complete medium. The minimal medium described is suitable to study spontaneous metabolic deficiciencies and regulation of enzyme activities inZ.mobilis.     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Could the improvements in the alcoholic fermentation of high glucose concentrations by yeast immobilization be explained by media supplementation?

Summary The maximal concentration of ethanol produced during the fermentation of 320 g/l glucose bySaccharomyces bayanus was higher when the yeast cells were immobilized either by adsorption on celite or by entrapment in k-carrageenan beads (from 10.5% with free cells up to 14.5% and 13.1% (v/v) respectively). This increase was due to medium supplementation with the compounds present in the immobilization supports.     

High level production of 5-aminolevulinic acid by Propionibacterium acidipropionici grown in a low-cost medium

5-Aminolevulinic acid (ALA) is an intermediate in the biosynthesis of tetrapyrroles. Its current production is expensive. We have developed a low-cost medium for Propionibacterium acidipropionici to produce extracellular ALA. When grown at 35?°C on a medium containing 3?% (w/v) food-grade sodium lactate supplemented with 18?g glycine/l, 4.05?g succinate/l, 1.8?g glucose/l, pH 7, it produced ALA up to 7.7?g/l over 6?days. Plant-growth promoting activity assays showed that the ALA was biologically active.     

Continuous production of phenylalanine using an Escherichia coli regulatory mutant

Summary Phenylalanine synthesis from glucose and ammonia was studied using a hyperproducing mutant of Escherichia coli. Kinetic parameters (typical values : 8.7 g phenylalanine/l, yield on glucose 0.19 g/g, productivity 0.44 g/l/h) were similar to batch culture values.     

Selective production of phenylalanine from phenylpyruvate using growing cells ofCorynebacterium glutamicum

Summary Phenylalanine was produced from phenylpyruvate by growing cells of a mutant strain ofCorynebacterium glutamicum over-producing valine. A defined medium was used to minimize the accumulation of valine. The maximum phenylalanine concentration achieved was 44.5 mM (7.5 g/l) with a phenylpyruvate molar conversion of 75%.     

Production of HBsAg by growth rate control with recombinantSaccharomyces cerevisiae in fed-batch

Summary To maintain a constant specific growth rate for a recombinantS.cerevisiae in fed-batch, the medium feeding rate has been controlled with respect to the hourly calculated glucose uptake rate. The recombinant yeast producing HBsAg showed the exponential production trend in proportion to the exponential cell growth. Total cell yield in fed-batch was about 0.402 g cells/g glucose, and HBsAg was produced about ten times more than in batch. Decrease of growth rate by HBsAg produced was not shown.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Response surface optimization ofLactobacillus plantarum batch growth

Summary Optimal conditions for batch growth ofLactobacillus plantarum, ATCC 8014, are a pH of 6.0, a temperature of 33°C, and an initial glucose concentration of 24 g/l. A maximum biomass concentration of 6.0 g/l was achieved. Growth parameters were also determined.     

Optimization of recombinant aminolevulinate synthase production in<Emphasis Type="Italic"> Escherichia coli</Emphasis> using factorial design

The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111. The effects of initial succinate, glucose and isopropyl--d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied. One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors. Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG. ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655. Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Production of ethanol and biomass from various carbohydrates byKluyveromyces fragilis

Summary Growth ofKluyveromyces fragilis NRC 2475 and the production of ethanol by the yeast were studied in the media containing one of the following sugars: glucose, lactose, galactose, or a glucose-galactose (50% 50%) mixture as a carbon source.The largest biomass yield and the lowest yield of ethanol were obtained in the medium containing glucose. The medium containing galactose gave the lowest yield of biomass and the largest yield of ethanol. When lactose was used for the growth and production of ethanol the obtained results for both biomass and ethanol were between those obtained with glucose and galactose.The ethanol productivities, expressed in terms of ethanol produced either per unit of cells, or per unit of cells and time, were the highest in the system with galactose and the lowest in that with glucose.     

Plant regeneration from leaf protoplasts of Solanum torvum

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10⁴ per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1–2 weeks and 4 weeks later p-calli were 1–3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CPA p-chlorophenoxyacetic acid - IAA indoleacetic acid - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthaleneacetic acid - 2ip 6-dimethylallyamino purine Michigan Agricultural Experiment Station Journal Article No. 12167     

Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

An investigation of ethanol inhibition and other limitations occurring during the fermentation of concentrated whey permeate byKluyveromyces fragilis

Based on the well-known fact thatKluyveromyces fragilis strains show sub-optimal performance when grown in concentrated whey permeate, previously optimized medium was investigated for possible limitations appearing at high concentrations. Shaken flask cultures showed that no additional vitamin or mineral sources were required when the optimized amount of yeast extract was added to the concentrated permeate. Several aspects of the ethanol inhibition of the growth ofK. fragilis NRRL 665 were investigated in continuous culture. The maximum ethanol concentration tolerated by this yeast, i.e. 45 g/l, was much lower than commonly reported for other strains. Ethanol and biomass production were also influenced by the increased ethanol concentration of the medium. At 31 g/l of alcohol product yield was reduced to 0.23 g/g whereas biomass yield was 0.05 g/g. Some evidence suggested that residence time and residual lactose concentration played a significant role in modulating the toxic effect of ethanol.     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

Use ofClostridium acetobutylicum P262 for production of solvents from whey permeate

Summary The production of solvents from whey permeate in batch fermentation usingClostridium acetobutylicum P262 was examined. An overall reactor productivity of 0.24 g/l.h was observed, representing a marked improvement over reports using other strains of clostridia. Using a semi-synthetic medium galactose was shown to be as effective a substrate as glucose. When whey permeate was used in which the lactose was hydrolysed prior to fermentation, preferential uptake of glucose over galactose was observed, and such hydrolysis provided no advantage to the fermentation process.     

Pantothenate limitation does not induce uncoupled growth of Zymomonas in chemostat culture

Summary Growth coefficients ofZymomonas mobilis were compared in glucose-limited chemostat culture using a complex medium and a defined minimal growth medium at non inhibitory concentrations of ethanol. Under carbon and energy limited conditions in the complex medium containing yeast extract, the max. molar growth yield (Y_G ^max) and maintenance energy coefficient (m_e) were 10.8 g cell/mol glucose and 8.3 mmol glu/g cell/hr, respectively. Glucose-limited growth in the minimal medium with NH₄Cl as nitrogen source promoted slight energetic uncoupling, as reflected in the decrease in the maximum growth yield. The growth yield with respect to calcium pantothenate was calculated to be 1.4×10⁴ g cell/g Ca-pantothenate. However, pantothenate-limited growth did not result in a decrease in growth yield nor an increase in the specific rate of glucose catabolism. Steady-state growth measurements failed to confirm the previously held view of Belaïchet al. (1972) that pantothenate deficiency induces energetic uncoupling inZymomonas.     

The effect of temperature,pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation

Summary Zymomonas mobilis, strain ATCC 10988, was used to evaluate the effects of pH (5.0 to 8.0), temperature (30°C to 40°C), and initial glucose concentration (75 g/l to 150 g/l) on the kinetics of ethanol production from glucose using batch fermentation. Specific ethanol production rate was maximum and nearly constant over a pH range of 6.0 to 7.5. End-of-batch ethanol yield and specific growth rate were insensitive to pH in the range of 5.0 to 7.5. End-of-batch ethanol yield was maximum and nearly constant between 30°C and 37°C but decreased by 24% between 37°C and 40°C. All other kinetic parameters are greatest at 34°C. End-of-batch ethanol yield is maximum at an initial glucose concentration of 100 g/l. Specific growth rate reaches a maximum at 75 g/l, but specific ethanol production rate decreases throughout the range. The optimum initial glucose concentration of 100 g/l gives the highest ethanol yield at a specific ethanol production rate less than 10% below the maximum observed.     

Growth ofCandida albicans in a minimal synthetic medium without biotin

Summary Growth ofCandida albicans strain B 311-10 was observed in a minimal synthetic biotin-free medium, using different glucose concentrations, during the first 30 hours of its development at 28 °C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherdet al. [12], with glucose (15 g/L) and biotin was modified: this vitamin was eliminated and the concentration of glucose was gradually lowered to 0.5 g/L. At 5 g/L of glucose and without biotin very good growth was obtained. Based on our results during the first 30 hours of growth, biotin has no influence on the yeast's growth. This medium would be useful for the study of the physiology ofC. albicans during the first period of its development.