A fast method for the quantification of methylamine in fermentation broths by gas chromatography

The objective of this study was to develop a method for the quantitative analysis of the methylamine concentration in fermentation broths of Hyphomicrobium zavarzinii ZV 580 cultures. For this purpose an established method for the quantification of free amino acids in such matrices was adapted and validated. The detection limit was 10 microM, the calibration curve showed good linearity (R2=0.9998) in the concentration range between 0.1 and 8 mM. The standard deviation of the injection-to-injection reproducibility (n=10) of the retention coefficient was <1%, that of the peak area<5%. In case of the sample-to-sample reproducibility (n=8), the standard deviation was <5% for the retention coefficient and <10% for the peak area. The validated method was successfully applied for monitoring a fed-batch bioprocess (starting volume: 8L, initial methylamine hydrochloride concentration: 10 mM) producing a dye-linked formaldehyde dehydrogenase in H. zavarzinii ZV 580.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Kinetics of CO conversion into H<Subscript>2</Subscript> by <Emphasis Type="Italic">Carboxydothermus hydrogenoformans</Emphasis>

The objective of this study was to improve the biological water–gas shift reaction for producing hydrogen (H₂) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L⁻¹. Results showed that the gas–liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g⁻¹ biomass VSS in order to avoid gas–liquid substrate transfer limitation. An average H₂ yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g⁻¹ VSS day⁻¹ were obtained at initial biomass densities between 5 and 8 mg VSS⁻¹. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g⁻¹ VSS day⁻¹ for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.     

An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain

A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l⁻¹) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol⁻¹ (0.92 g g⁻¹) and 0.29 mol mol⁻¹ (0.10 g g⁻¹), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.     

Acid phosphatase production by recombinant <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g⁻¹ DYB) and laboratory fermenter (18,465 U g⁻¹ DYB), respectively.     

Optimization of culture conditions and scale-up to pilot and plant scales for coenzyme Q<Subscript>10</Subscript> production by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

This report describes the optimization of culture conditions for coenzyme Q₁₀ (CoQ₁₀) production by Agrobacterium tumefaciens KCCM 10413, an identified high-CoQ₁₀-producing strain (Kim et al., Korean patent. 10-0458818, 2002b). Among the conditions tested, the pH and the dissolved oxygen (DO) levels were the key factors affecting CoQ₁₀ production. When the pH and DO levels were controlled at 7.0 and 0–10%, respectively, a dry cell weight (DCW) of 48.4 g l⁻¹ and a CoQ₁₀ production of 320 mg l⁻¹ were obtained after 96 h of batch culture, corresponding to a specific CoQ₁₀ content of 6.61 mg g-DCW⁻¹. In a fed-batch culture of sucrose, the DCW, specific CoQ₁₀ content, and CoQ₁₀ production increased to 53.6 g l⁻¹, 8.54 mg g-DCW⁻¹, and 458 mg l⁻¹, respectively. CoQ₁₀ production was scaled up from a laboratory scale (5-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V _tip) as a scale-up parameter. CoQ₁₀ production at the laboratory scale was similar to those at the pilot and plant scales. This is the first report of pilot- and plant-scale productions of CoQ₁₀ in A. tumefaciens.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Production ecology of phyto- and zooplankton in a eutrophic pond dominated byChaoborus flavicans (Diptera: Chaobolidae)

Primary production of phytoplankton and secondary production of a daphnid and a chaoborid were studied in a small eutrophic pond. The gross primary production of phytoplankton was 290 gC m⁻² per 9 months during April–December. Regression analysis showed that the gross primary production was related to the incident solar radiation and the chlorophylla concentration and not to either total phosphorus or total inorganic nitrogen concentration. The mean chlorophylla concentration (14.2 mg m⁻³), however, was about half the expected value upon phosphorus loading of this pond. The mean zooplankton biomass was 1.60 g dry weight m⁻², of whichDaphnia rosea and cyclopoid copepods amounted to 0.69 g dry weight m⁻² and 0.61 g dry weight m⁻², respectively. The production ofD. rosea was high during May–July and October and the level for the whole 9 months was 22.6 g dry weight m⁻².Chaoborus flavicans produced 10 complete and one incomplete cohorts per year. Two consecutive cohorts overlapped during the growing season. The maximum density, the mean biomass, and the production were 19,100 m⁻², 0.81 g dry weight m⁻², and 11.7 g dry weight m⁻²yr⁻¹, respectively. As no fish was present in this pond, the emerging biomass amounted to 69% of larval production. The production ofC. flavicans larvae was high in comparison with zooplankton production during August–September, when the larvae possibly fed not only on zooplankton but also algae.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

Heterotrophic high-cell-density fed-batch and continuous-flow cultures of <Emphasis Type="Italic">Galdieria sulphuraria</Emphasis> and production of phycocyanin

Production of biomass and phycocyanin (PC) were investigated in highly pigmented variants of the unicellular rhodophyte Galdieria sulphuraria, which maintained high specific pigment concentrations when grown heterotrophically in darkness. The parental culture, G. sulphuraria 074G was grown on solidified growth media, and intensely coloured colonies were isolated and grown in high-cell-density fed-batch and continuous-flow cultures. These cultures contained 80–110 g L⁻¹ biomass and 1.4–2.9 g L⁻¹ PC. The volumetric PC production rates were 0.5–0.9 g L⁻¹ day⁻¹. The PC production rates were 11–21 times higher than previously reported for heterotrophic G. sulphuraria 074G grown on glucose and 20–287 times higher than found in phototrophic cultures of Spirulina platensis, the organism presently used for commercial production of PC.     

Photoautotrophic Production of Lipids by Some <Emphasis Type="Italic">Chlorella</Emphasis> Strains

The microalgae Chlorella protothecoides UTEX 25, Chlorella sp. TISTR 8991, and Chlorella sp. TISTR 8990 were compared for use in the production of biomass and lipids under photoautotrophic conditions. Chlorella sp. TISTR 8990 was shown to be potentially suitable for lipid production at 30°C in a culture medium that contained only inorganic salts. For Chlorella sp. TISTR 8990 in optimal conditions in a stirred tank photobioreactor, the lipid productivity was 2.3 mg L⁻¹ h⁻¹ and after 14 days the biomass contained more than 30% lipids by dry weight. To attain this, the nitrogen was provided as KNO₃ at an initial concentration of 2.05 g L⁻¹ and chelated ferric iron was added at a concentration of 1.2 × 10⁻⁵ mol L⁻¹ on the ninth day. Under the same conditions in culture tubes (36 mm outer diameter), the biomass productivity was 2.8-fold greater than in the photobioreactor (0.125 m in diameter), but the lipid productivity was only 1.2-fold higher. Thus, the average low-light level in the photobioreactor actually increased the biomass specific lipid production compared to the culture tubes. A light-limited growth model closely agreed with the experimental profiles of biomass production, nitrogen consumption, and lipid production in the photobioreactor.     

The effect of phytohormones on growth and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

Summary Few studies have focused on the effect of a broad range of phytohormones on growth and secondary metabolism of a single hairy root species. We measured growth, development, and production of the antimalarial drug, artemisinin, in Artemisia annua hairy roots in response to the five main hormones: auxins, cytokinins, ethylene, gibberellins (GA), and abscisic acid (ABA). Single roots grown in six-well plates in medium B5 with 0.01 mgl⁻¹ (0.029 μM) GA₃ produced the highest values overall in terms of the number of lateral roots, length of the primary root, lateral root tip density, total lateral root length, and total root length. When the total root lengths are compared, the best conditions for stimulating elongation appear to be: GA 0.01 mgl⁻¹ (0.029μM)> ABA 1.0 mgl⁻¹ (3.78μM)=GA 0.02 mgl⁻¹ (0.058μM). Bulk yields of biomass were inversely proportional to the concentration of each hormone tested. All cultures provided with ABA yielded the highest amount of biomass. Both 6-benzylaminopurine and 2-isopentenyladenine inhibited root growth, however, only 2-isopentenyladenine stimulated artemisinin production, more than twice that of the B5 controls, and more than any other hormone studied. These results will prove useful in increasing hairy root growth and artemisinin production.     

Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Enhanced iturin A production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on suppression of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.     

Strain improvement of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C-30 for increased cellulase production

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml⁻¹, a CMCase activity of 54.2 U ml⁻¹, a β-glucosidase activity of 0.39 U ml⁻¹, and a fungal biomass of 12.6 mg ml⁻¹. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml⁻¹). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.