Effect of perfluorodecalin as an oxygen carrier on actinorhodin production by Streptomyces coelicolor A3(2)

Perfluorodecalin, a perfluorocarbon (PFC), was used in this investigation as a dissolved oxygen carrier in the media of Streptomyces coelicolor cultures. The effects of different concentrations of PFC, PFC emulsified with pluronic F-68 and pluronic alone were investigated in the shake-flask cultures using both defined and complex media. In the defined medium with PFC alone, the maximum biomass and actinorhodin concentrations and the volumetric substrate consumption rates increased with increasing PFC concentration. They decreased dramatically, however, when the PFC concentration exceeded 50% (v/v). Emulsifying the PFC with pluronic F-68 resulted in a significant increase in antibiotic concentration while growth was unaffected. The inclusion of more than 4 g/l pluronic alone in the fermentation medium inhibited the growth. In the complex medium with 40% (v/v) PFC, although the final antibiotic concentration was unaffected, the onset of actinorhodin accumulation was 2 days earlier than that in the control. It was demonstrated that PFC and emulsified PFC did not have any deleterious effects on S. coelicolor cultures.     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

Effects of perfluorinated oxygen carrier application in yeast, fungi and plant cell suspension cultures

The growth of the yeast Saccharomyces cerevisiae, the fungus Rhizopus nigricans and Nicotiana tabacum cells with perfluorodecalin as an oxygen carrier has been studied. The volumetric mass transfer coefficient (k_La) measured by the dynamic method was higher for the perfluorodecalin oxygenation system than for the conventional aeration system. The results show that perfluorocarbon can be successfully used as an efficient gas carrier, especially for the culture of delicate plant cells. The increase in yeast biomass in the suspension culture aerated by perfluorodecalin was as much as 110% higher than in the culture aerated by air. The fungus R. nigricans grew better when the conventional aeration system was used due to the fact that growth of the mycelium is limited by the transport of oxygen by diffusion in the pellets rather than by interfacial oxygen transport. In the case of isolated tobacco cells, an increase of over 350% in biomass growth was observed for the PFC aeration system.     

The effect of oxygen concentration on photosynthetic biomass production by algae

The effect of decreased oxygen concentration on photosynthetic biomass production was determined for Euglena gracilis Klebs strain z and Chlamydomonas reinhardtii Dangeard. At a constant carbon dioxide concentration of 0.03% (v/v), decreasing the oxygen concentration from 21% to 2% (v/v) gave a two-fold increase in dry-weight yield for E. gracilis; a result consistent with the operation of a functional glycollate pathway in this alga. A similar effect of oxygen concentration on dry-weight yield was not observed with C. reinhardtii.     

Microalgal system for treatment of effluent from poultry litter anaerobic digestion

The potential of mixotrophic microalgae to utilize poultry litter anaerobic digester (AD) effluent (PLDE) as nutritional growth medium was evaluated. Three algal strains viz. Chlorella minutissima, Chlorella sorokiniana and Scenedesmus bijuga and their consortium showed significant biomass productivity in 6% (v/v) concentration of PLDE in deionized water. Multiple booster dosage of PLDE supported better growth relative to a single dose PLDE. The maximum biomass productivity of 76 mg L⁻¹ d⁻¹ was recorded. The biomass was rich in protein (39% w/w) and carbohydrates (22%) while lipids (<10%) were low, making it most suitable as an animal feed supplement. The mixotrophic algae showed sustainable growth against variations in PLDE composition in different AD batches, thus proving to be a suitable candidate for large scale wastewater treatment with concomitant production of renewable biomass feedstock for animal feed and bioenergy applications.     

Investigation of biomass concentration,lipid production,and cellulose content in Chlorella vulgaris cultures using response surface methodology

The microalgae Chlorella vulgaris produce lipids that after extraction from cells can be converted into biodiesel. However, these lipids cannot be efficiently extracted from cells due to the presence of the microalgae cell wall, which acts as a barrier for lipid removal when traditional extraction methods are employed. Therefore, a microalgae system with high lipid productivity and thinner cell walls could be more suitable for lipid production from microalgae. This study addresses the effect of culture conditions, specifically carbon dioxide and sodium nitrate concentrations, on biomass concentration and the ratio of lipid productivity/cellulose content. Optimization of culture conditions was done by response surface methodology. The empirical model for biomass concentration (R² = 96.0%) led to a predicted maximum of 1123.2 mg dw L^?1 when carbon dioxide and sodium nitrate concentrations were 2.33% (v/v) and 5.77 mM, respectively. For lipid productivity/cellulose content ratio (R² = 95.2%) the maximum predicted value was 0.46 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 when carbon dioxide concentration was 4.02% (v/v) and sodium nitrate concentration was 3.21 mM. A common optimum point for both variables (biomass concentration and lipid productivity/cellulose content ratio) was also found, predicting a biomass concentration of 1119.7 mg dw L^?1 and lipid productivity/cellulose content ratio of 0.44 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 for culture conditions of 3.77% (v/v) carbon dioxide and 4.01 mM sodium nitrate. The models were experimentally validated and results supported their accuracy. This study shows that it is possible to improve lipid productivity/cellulose content by manipulation of culture conditions, which may be applicable to any scale of bioreactors. Biotechnol. Bioeng. 2013; 110: 2114–2122. © 2013 Wiley Periodicals, Inc.     

Citric acid production by Yarrowia lipolytica: Effect of nitrogen and biomass concentration on yield and productivity

Summary Citric acid yields of Yarrowia lipolytica (NRRL Y-1095) grown on glucose ranged from 0.38–0.77 g/g and were dependent on both biomass and nitrogen concentration (as NH₄Cl and yeast extract). Increasing the biomass concentration by 3% (w/v) increased fermentor productivities from 0.6 to 1.22 g citric acid/L h.     

Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalgaPhaeodactylum tricornutum following a three-step process: fatty acid extraction by direct saponification of wet biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds and EPA isolation by preparative HPLC. Direct saponification of wet biomass was carried out with KOH-ethanol (96% v:v) (1 h, 60 °C), extracting 91% of the EPA. PUFAs were concentrated by the urea method with an urea/fatty acid ratio of 4:1 at a crystallization temperature of 28 °C using methanol as the urea solvent. An EPA concentration ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PUFA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C₁₈, 4.7 cm i.d. × 30 cm) and methanol-water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in theP. tricornutum biomass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PUFA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d^–1). Finally, a preliminary cost statement has been calculated.     

Transport of nitric oxide by perfluorocarbon emulsion

Perfluorocarbon (PFC) emulsions can transport and release various gases based on concentration gradients. The objective of this study was to determine the possibility of carrying and delivering exogenous nitric oxide (NO) into the circulation by simply loading PFC emulsion with NO prior infusion. PFC was equilibrated with room air (PFC) or 300 ppm NO (PFC‐NO) at atmospheric pressure. Isotonic saline solution was used as a volume control (Saline). PFC and PFC‐NO were infused at a dose of 3.5 mL/kg in the hamster window chamber model. Blood chemistry, and systemic and microvascular hemodynamic response were measured. Infusion of PFC preloaded with NO reduced blood pressure, induced microvascular vasodilation and increased capillary perfusion; although these changes lasted less than 30 min post infusion. On the other hand, infusion of PFC (without NO) produced vasoconstriction; however, the vasoconstriction was followed by vasodilatation at 30 min post infusion. Plasma nitrite and nitrate increased 15 min after infusion of NO preloaded PFC compared with PFC, 60 min after infusion nitrite and nitrate were not different, and 90 min after infusion plasma S‐nitrosothiols increased in both groups. Infusion of NO preloaded PFC resulted in acute vascular relaxation, where as infusion of PFC (without NO) produced vasoconstriction, potentially due to NO sequestration by the PFC micelles. The late effects of PFC infusion are due to NO redistribution and plasma S‐nitrosothiols. Gas solubility in PFC can provide a tool to modulate plasma vasoactive NO forms availability and improve microcirculatory function and promote increased blood flow. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1565–1572, 2013     

虎掌菌菌丝富硒培养的研究

为生产合适的硒源提供一种思路,以菌丝体生物量、含硒量、还原糖、氨态氮和蛋白质为指标,采用四因素三水平正交设计法优化虎掌菌的富硒发酵条件,探讨不同浓度的硒对虎掌菌固体培养菌丝生长和液体培养产生的生物组分的影响。结果表明,高浓度的硒抑制虎掌菌菌丝体生长;正交试验选择不同的衡量指标,由极差分析得出各因素的影响程度大小,结合证实试验得到以还原糖为指标的最优组合为葡萄糖6%(质量分数)、酵母浸膏3%(质量分数)、KH_2PO_4 0.1%(质量分数)、Na_2SeO_3 0.6 mmol/L,其菌丝体生物量和氨态氮含量较高。与对照相比,加硒后虎掌菌发酵液中氨态氮、还原糖和总糖含量显著增加(P0.05);当硒浓度为0.5 mmol/L时,氨态氮、还原糖和总糖含量均达到最高值。菌丝体生物量和可溶性蛋白质分别在硒浓度为0.2 mmol/L和0.4 mmol/L时达到最大值。虎掌菌富硒培养后,发酵液的营养成分含量会发生变化。     

Production of microalgae from olive mill wastewater

With olive mill wastewater (alpechin) used as a nutrient medium, the influence of the aeration level and the composition of the culture medium were analysed in relation to the concentration of alpechin and KNO₃ added in a batch culture of the microalgae Chlorella pyrenoidosa and Scenedesmus obliquus. The pH and temperature of the culture were previously fixed at 6.5 and 30°C. As kinetic parameters, the maximum specific growth rate (μ_m) and the biomass productivity (P_max) were determined. The biomass composition obtained was evaluated for chlorophyll and crude protein content as well as fatty-acid composition corresponding to the lipid fraction. To achieve a balanced biomass composition in terms of proteins and lipids, the most suitable conditions included an alpechin concentration of 10%, without the addition of nitrates and with an aeration level of 1 v/v min. Under these conditions, the μ_m values reached with both microalgae were around 0.03h⁻¹.     

Improved production of citric acid by Yarrowia lipolytica using oleic acid as the oxygen‐vector and co‐substrate

Yarrowia lipolytica is able to secrete large amounts of citric acid (CA), which is greatly affected by the dissolved oxygen concentration (DOC) in the fermentation medium. In this study, oleic acid was selected as oxygen‐vector to improve DOC during CA fermentation. When 2% (v/v) of oleic acid was added to the culture broth, higher DOC (>42.1%) was determined throughout the CA synthesis phase. The yield of CA reached a maximum of 32.1 g/L (25.4% higher than the control) and the biomass was 8.8 g/L. The substrate uptake rate, products formation rate and key enzyme activities were also determined, and the results indicated that CA synthesis was strengthened with oleic acid addition. Furthermore, it was detected that oleic acid could be assimilated by the cells, which means that oleic acid could be served both as oxygen‐vector and co‐substrate for CA synthesis by Y. lipolytica. In a bioreactor with working volume of 3 L, the highest concentration of CA reached to 36. 4 g/L in the presence of 2% (v/v) oleic acid after 192 h of fermentation. These results confirmed that oleic acid could be applied in the large‐scale production of CA by Y. lipolytica.     

Nitrate reduction and nitrate content in ash trees (Fraxinus excelsior L.): distribution between compartments,site comparison and seasonal variation

Summary Nitrate reductase activity (NRA), nitrate content and biomass components of leaflets, leaf stalks, old stem, current-year stem and roots of ash trees (Fraxinus excelsior L.) growing in their natural habitats were investigated. In addition, NRA, total nitrogen and nitrate concentration were analyzed in the leaves and roots of ash trees from four different field sites. The highest NRA per gram biomass and also per total compartment biomass was found in the leaflets, even though root biomass was much higher than total leaflet biomass. The highest nitrate concentrations were found in the leaf stalks. Correlations between nitrate availability in the soil and NRA in leaves were not significant due to high variability of the actual soil nitrate concentrations. The seasonal variation in foliar NRA, nitrate concentration and total nitrogen concentration is much smaller in F. excelsior than reported for herbaceous species and is mainly caused by changes in the actual soil nitrate availability and by senescence of the leaves.     

Study of docosahexaenoic acid production by the heterotrophic microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> CCMP 316 using carob pulp as a promising carbon source

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.     

Sucrose-induced osmotic stress affects biomass,metabolite, and antioxidant levels in root suspension cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

In this study, we investigated the influence of initial sucrose concentration on the accumulation of biomass, phenols, flavonoids, chlorogenic acid, and hypericin in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium, 1.0 mg l⁻¹ indolebutyric acid (IBA), 0.1 m g l⁻¹ kinetin, and different concentrations 0, 1, 3, 5, 7, or 9% in w/v) of sucrose and were maintained in darkness. The medium supplemented with 3% (w/v) sucrose resulted in the optimum biomass accumulation, but higher sucrose concentrations (5, 7, and 9%) inhibited biomass accumulation due to the relatively higher osmotic pressure. However, the amount of total phenols, flavonoids, chlorogenic acid, and total hypericin was increased with the roots grown in the medium supplemented with 5, 7, and 9% (w/v) sucrose. The antioxidant potential of methanolic extract [1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) radical scavenging activities] of H. perforatum adventitious roots was also assessed and correlated with the metabolite accumulation. Cultures maintained with higher initial sucrose concentration (5, 7, and 9% w/v) showed increased accumulation of phenols, flavonoids, chlorogenic acid, and total hypericin, and this might be due to the osmotic stress at elevated sucrose concentrations. To verify the effect of osmotic stress on lipid peroxidation, the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and proline were determined in the adventitious roots and the results revealed a marked increase in the concentrations of these compounds. These results suggest that optimal adventitious root biomass could be achieved in the MS medium with 3% (w/v) sucrose and increased sucrose concentration resulted in osmotic stress and, in turn, induces the accumulation of secondary metabolites.     

Elemental composition of two coexisting Daphnia species during the seasonal course of population development in Lake Constance

Summary In 1986 and 1987 the seasonal course of population density, life history traits and biomass composition (CHN-elemental and ash) of two coexisting Daphnia species from L. Constance (Überlinger See) were studied. The two daphnids are morphologically similar, but demonstrate different seasonal variation of behaviour (i.e. diurnal vertical migration). With the exception of nitrogen content, the mean biomass composition of adults and that of eggs were significantly different: the dry biomass of eggs (adults) contained on average 55.6 (46.1) % C, 7.9 (6.5) % H, 9.7 (9.7) % N, and 10.0 (23.8) % ash. These values were similar to those of other daphnids; it may be concluded that the average daphnid composition is not species specific, but is determined mainly by the position in the life cycle and by the nutritional state of the animal. A distinct seasonal variation in the elemental composition of the biomass of adult daphnids was observed. It related significantly to the concentration of food, as indicated by the epilimnetic chlorophyll-a concentrations and by Secchi depth. From the amplitude of seasonal variation of elemental composition and from the shape of relationships between the elemental composition of daphnids and their food, it can be concluded that in summer the non-migrating D. galeata lives under stronger food limitation than the migrating H. hyalina. In spring, there were no differences in the biomass composition of either species. In summer, the adult fcmales of D. galeata contained significantly less carbon, and hydrogen than those of D. hyalina. In most parameters of biomass (i.e. biomass composition of adults) and of life history (i.e. clutch size, mean adult weight), D. galeata displayed higher coefficients of variation. A tendency in D. galeata to have peaks of population development carlier in the season and in D. hyalina to have them towards autumn was observed. These contrasting responses of the populations development, the seasonal variability of the biomass composition seem to support the view of Geller (1986) who called D. galeata and D. hyalina exploitative and conservative strategists respectively.This research was supported by the Deutsche Forschungsge-meinschaft within the Sonderforschungsbereich Cycling of Matter in Lake Constance (SFB 248), and by scholarship to R.B. from the Deutscher Akademischer Austauschdienst (DAAD)     

Microcalorimetry as a diagnostic and analytical tool for the assessment of biodegradation of 2,4-D in a liquid medium and in soil

The biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) byPseudomonas cepacia was assessed by microcalorimetry in a liquid medium and in sterilized soil at 25°C under aerobic conditions. It was found that thermograms of the rate of heat evolved versus time (dQ/dt versust) can be used as a diagnostic tool to identify the timet ₁ required for the primary biodegradation of 2,4-D and the timet _f required for the completion of the biodegradation activity in a liquid medium as well as in soil. Microcalorimetry can also be used as an analytical tool to monitor the progress of 2,4-D consumption during the biodegradation process in a liquid medium and to measure the importance of the soil sorption/desorption of intermediate metabolites. A new concept called bioeffort was defined as the product of the biodegradation time (t) and the biomass concentration (X) at that time. This concept was used to predict either the biomass concentration required or the duration of the primary biodegradation of 2,4-D in soil from the data obtained from a liquid medium.     

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent (ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg⁻¹ with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.     

Single-cell protein of Rhodotorula sp. Y-38 from ethanol, acetic acid and acetaldehyde

Summary Continuous cultivation of Rhodotorula sp. Y-38 was carried out on ethanol, acetic acid or acetaldehyde. At a feed concentration of 1.0 % (w/v) ethanol, the cell yield of 64 g/100 g ethanol and crude protein of 52 g/100 g biomass were obtained at D=0.5 h^-1. The respective value of the content of amino acids and nucleic acids was 42.6 and 9.4 g/100 g biomass. At 2.0 % (w/v) acetic acid, cell yield was found to be 50 g/100 g acetic acid at D=0.4 h^-1. The optimum dilution rate ranged between 0.3 and 0.4 h^-1. At 0.05 % (w/v) acetaldehyde, the maximum cell yield was obtained at D=0.14 h^-1.     

Carbon dioxide-gassed fluorocarbon enhances micropropagation of rose (Rosa chinesis Jacq.)

The inert perfluorochemical (PFC) liquid, perfluorodecalin (Flutec PP6), has been used to increase the CO₂ supply to cultured shoots of Rosa chinensis Jacq. cv. Baby Love. Culture of shoots in semi-solid medium overlaying CO₂-gassed PFC (2 mbar; 5 min repeated every 7 days) for up to 42 days, increased biomass as reflected by significant (P<0.01) increases in shoot number, number of leaves per shoot and mean shoot fresh weight. Additionally, there were significant (P<0.01) increases in the number of roots and their fresh and dry weights following a further 10 days of culture on rooting medium prior to transfer of plants to the glasshouse. Treatment of cultured rose shoots with CO₂-gassed PFC also significantly reduced (P<0.01) the accumulation of phenolic compounds in roots. The total chlorophyll of aerial parts was unaffected, although total protein in shoots and roots was significantly (P<0.01) lower than in the control. The biotechnological implications of this novel cultural régime are discussed for the micropropagation of woody species. Received: 5 June 1996 / Revision received: 29 July 1996 / Accepted 19 August 1996