Reactive extraction of cephalosporin antibiotics in hollow fiber membrane

Non-dispersive reactive extraction of cephalosporin antibiotics has been studied using hollow fiber membrane modules. Extraction as well as stripping has been studied using a pH swing procedure. Cephalosporin was extracted from an aqueous solution of cephalosporin having a pH above the pK_a2 value to an organic phase containing Aliquat-336 as the extractant and n-heptane as the diluent. The solute was stripped from the loaded organic phase to another aqueous phase of pH maintained well below the pK_a2 value of the cephalosporin. The extraction cum stripping relies on pH dependance of the distribution coefficient of cephalosporin in aqueous phase. Reasonably high solute recovery and mass transfer rate have been achieved in the hollow fiber module. Mass transfer performance of a single module has been evaluated and experimentally observed low value of height of transfer unit (HTU) indicates good prospect of hollow fiber membrane for the extraction duty.     

On liquid-liquid mass transfer in two-liquid-phase fermentations

Almost all two-liquid phase bioprocesses are characterized by the presence of surface active materials (biosurfactants), which significantly influence the interaction between the phases. In order to predict mass transfer rates during cultivations of Pseudomonas oleovorans biosurfactant was isolated from the biosuspension and added in defined amounts to n-octane/water model-dispersions. Effects of biosurfactant concentration on interfacial tension, mean Sauter-diameter, drop size distribution, dispersion stability and liquid-liquid mass transfer coefficients were studied. A comparison was made between calculated solvent transfer rates (STR) and measured solvent uptake rates (SUR) of P. oleovorans cultures. With increasing interfacial surfactant concentration interfacial tension and mean Sauter-diameter decreased until a minimum for both, interfacial tension and mean Sauter-diameter, were reached. Interfacial tension measurements indicate that these minima have to be attributed to a maximum monomolecular surfactant concentration and the formation of polymolecular adsorption layers. Drop size distributions showed that, coalescence and droplet break-up disappear because droplets are stabilized by the biosurfactant adsorption layers at the interface. Mass transfer regime shifted from forced convection and surface renewal to diffusion. Comparison of solvent uptake rates (SUR) and solvent transfer rates (STR) showed that n-octane transfer usually will not be limiting P. oleovorans cultures, however, can become dominant in cultures where solvents with very low miscibilities like n-decane are used.     

Biocatalytic membranes for ultrafiltration treatment of wastewater containing dyes

A possibility to prepare the biofunctional membranes showing the biocatalytic properties and use those in post-treatment of wastewater containing synthetic dyes have been established. Selected Pseudomonas mendocina and Bacillus subtilis cultures were used as biocatalysts for dye destruction. It has been established that cells in spore form are able to survive in N-methylpyrrolidone that allow to use method of polymer solution casting for membrane preparation. The optimal conditions for entrapping of whole cells of microorganisms into the polymer matrix have been determined. Membrane biocatalytic activity has been studied depending on method of casting solution preparation, biocatalyst loading and operating parameters. Dye destruction occurs both in membrane pores and on membrane surface. Membrane obtained provide discolouring of treated solutions (permeate). The dye concentration in retentate depends on the trans-membrane fluxes. The concentration in retentate need not be observed at relatively low fluxes (up to 20 l/m² h).     

Two-stage recovery of S-adenosylmethionine using supported liquid membranes with strip dispersion

We report the selective recovery of S-adenosylmethionine (SAM) from fermentation broths using a two-stage supported liquid membrane system with strip dispersion (SLM-SD). The system utilized two MiniModule^® hollow-fiber membrane modules as microporous supports and an organic membrane solution consisting of the extractants of sodium di-2-ethylhexyl sulfosuccinate (AOT), di-(2-ethylhexyl)phosphoric acid (DEHPA), and trioctylphosphine oxide (TOPO) in the solvent n-octanol. SAM was extracted in the first membrane module. Methionine (Met) was captured by the first stripping solution and further purified in the second membrane module. pH values in the feed phase and the first and second stripping solutions, extractant concentrations, NaCl concentration, and the SAM acceptor in the first stripping solution were optimized. Strip dispersion mixing speed, pressure differences across the membranes, and flow rates of the feed and strip dispersion phases were investigated experimentally. The optimal extractant concentrations were: AOT 2.78 wt%, DEHPA 27.0 wt%, and TOPO 1.61 wt%. The optimal pH values in the feed phase and the first and second stripping solution were 3.0, 2.5, and 1.0, respectively. SAM extraction efficiency of 98.7%, SAM recovery efficiency of 91.8% and Met removal efficiency of 85.4% were achieved within 5 h. Finally, the mass transfer analysis indicated that the mass transfer resistances from the extraction reaction and the membrane phase were predominant.     

Lipid biomarkers indicate different ecological niches and trophic relationships of the Arctic hyperiid amphipods Themisto abyssorum and T. libellula

The hyperiid amphipods Themisto libellula and T. abyssorum are important components of Arctic pelagic ecosystems. Both species are carnivorous and prey on mesozooplankton. They represent a substantial food source for marine vertebrates and are a key link between zooplankton secondary production and higher trophic levels. We present data on the total lipid content, lipid class and fatty acid composition of T. libellula and T. abyssorum from northern Fram Strait and the central Arctic Ocean. Both species had moderate to high lipid contents of 14-42% of body dry mass. In T. abyssorum, total lipid content was correlated to body mass, while T. libellula showed sex-related differences in lipid content. Despite their smaller body size, females of T. libellula had higher lipid contents than males. Wax esters represented the major lipid class in both species with 41-43% of total lipid, while triacylglycerols contributed 23-32%. The fatty acid composition was dominated by the long-chain polyunsaturated moieties 20:5(n-3) and 22:6(n-3), short-chain saturated compounds (16:0 and 14:0) and monounsaturated fatty acids of varying length, i.e. 16:1(n-7), 20:1(n-9), 18:1(n-9) and 22:1(n-11). Species-specific and geographic variations in the fatty acid and alcohol patterns were apparently linked to differences in diet and life-cycle. High amounts of the fatty acids and alcohols 20:1(n-9) and 22:1(n-11) in T. libellula indicate predation on herbivorous Calanus copepodids. In addition, elevated levels of 20:5(n-3) in T. libellula indicate a close connection with ice-algal production and the importance of cryo-pelagic coupling processes (i.e. exchange processes between the sea ice and the pelagic communities) for the nutrition of this high-Arctic epipelagic species. In contrast, T. abyssorum is characterised by lower amounts of 20:5(n-3) and its biomarker ratios indicate a higher trophic level. This observation is consistent with the subarctic-boreal origin of T. abyssorum and its occurrence in deeper layers of the Arctic Ocean, where it may feed on omnivorous and/or carnivorous prey.     

Simultaneous extraction and concentration of penicillin G by hollow fiber renewal liquid membrane

In this article, hollow fiber renewal liquid membrane (HFRLM) technique was used for recovery of penicillin G from aqueous solution. The organic solution of 7 vol % di‐n‐octylamine (DOA) + 30 vol % iso‐octanol + kerosene was used as liquid membrane phase, and Na₂CO₃ aqueous solution was used as stripping phase. Experiments were performed as a function of carrier concentration in the organic phase, organic/aqueous volume ratio, pH, and initial penicillin G concentration in the feed phase, pH in the stripping phase, flow rates, etc. The results showed that the HFRLM process was stable and could carry out simultaneous extraction and concentration of penicillin G from aqueous solutions. As a carrier facilitated transport process, the addition of DOA in organic phase could greatly enhance the mass transfer rate; and there was a favorable organic/aqueous volume ratio of 1:20 to 1:30 for this system. The mass transfer flux and overall mass transfer coefficient increased with decreasing pH in the feed phase and increasing pH in the stripping phase, because of variation of the mass transfer driving force caused by pH gradient and distribution equilibrium. The flow rate of the shell side had significant influence on the mass transfer performance, whereas the effect of flow rate of lumen side on the mass transfer performance was slight because of the mass transfer intensification of renewal effect in the lumen side. The results indicated that the HFRLM process was a promising method for the recovery of penicillin G from aqueous solutions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Transformed T0 orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds

A highly efficient and reproducible transformation system for orchardgrass (Dactylis glomerata L. cv. Rapido, 2n=42=28) was established using microprojectile bombardment of highly regenerative, green tissues derived from mature seeds. These tissues, induced from embryogenic callus, were bombarded with a mixture of three plasmids containing the hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and #-glucuronidase (uidA; gus) genes. From 147 individual explants bombarded, 11 independent hygromycin-resistant lines (7.5%) were obtained after an 8- to 16-week selection period using 30-50 mg/l hygromycin B. Of the 11 independent lines, ten (91%) were regenerable. The presence and integration of the transgene(s) were assessed using PCR and DNA blot hybridization. Coexpression frequency of the three transgenes (hpt/bar/uidA) in T₀ plants was 20%, and of two transgenes, either hpt/bar or hpt/uidA, 45-60%. Due to greenhouse conditions optimized for the growth of other species, T₁ seed has not been obtained from these plants. While the inability to analyze progeny plants precludes the conclusive demonstration of stable transformation, the results of all molecular and biochemical analyses of T₀ plants are consistent with the production of stably transformed plants. Frequent change in ploidy level was observed in transformed T₀ orchardgrass plants. Plants from only three of the ten independent lines analyzed had the normal tetraploid number of chromosomes (2n=42=28), while plants from seven lines (70%) were octaploid (2n=82=56). The octaploid plants had abnormal morphological features, such as narrower, thicker and more upright leaves.     

Equilibrium studies on reactive extraction of succinic acid from aqueous solutions with tertiary amines

Reactive extraction of succinic acid from aqueous solutions with various tertiary amines dissolved in 1-octanol and n-heptane has been studied as a function of the acid concentration and the chain length of tertiary amine. When 1-octanol was used as diluent, the extractability of the tertiary amine was proportional to the chain length of tertiary amine at the same concentration of amines. The loading values were also proportional to the chain length of tertiary amine. However, when n-heptane was used as diluent, the extractability and loading values of the tertiary amine were inversely proportional to the chain length of amines. It was due to the aggregate formation of the acid-amine complex.     

Agrobacterium-mediated transformation of Campanula glomerata

A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg/l vancomycin and 100 mg/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of A. tumefaciens, LBA4404 or EHA105, both of which harbored the binary vector pGUSINT, coding for the uidA and neomycin phosphotransferase II (nptII) genes. While the transformation frequency (2-3%) was similar for both strains, A. tumefaciens LBA4404 was effectively eliminated from Campanula at a lower concentration of antibiotic as compared to EHA105. The concentration of individual antibiotics required to eliminate EHA105 resulted in a decreased rate (55-67%) of regeneration. The highest percentage of explants that regenerated plants (79%) and the highest regeneration rate was achieved with 100 mg/l cefotaxime combined with 400 mg/l vancomycin. Plants were also transformed with the isopentenyl transferase (ipt) gene using LBA4404 containing the 35S-ipt vector construct (pBC34).     

Effects of operating parameters on performances of nitrification and denitrification processes

Nitrification and denitrification of synthetic wastewater was studied by using two reactors in series. An activated sludge unit was used for nitrification followed by a downflow biofilter (packed column) for denitrification. A glucose solution was fed to the denitrification column to supply carbon source. Effects of important process variables such as sludge age, hydraulic residence time and feed ammonium concentration on system's performance were investigated. Effluent ammonium-nitrogen (NH4-N) concentration decreased with increasing sludge age and hydraulic residence time and remained constant for sludge age and hydraulic residence times greater than 12 d and 15 h, respectively. Feed ammonium-nitrogen concentration above 200 mg/l resulted in significant levels of NH4-N in the effluent at Š_c = 15 d and Š_H = 12 h in nitrification. Performance of denitrification stage was not satisfactory for feed NO₃-N concentrations above 150 mg N/l resulting in significant effluent NO₃-N levels at hydraulic residence time of Š_H = 6 h.     

Transgenic plants of blue grama grass,Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells

For the first time, the production of transgenic plants of the forage grass blue grama, Bouteloua gracilis [H.B.K.] Lag. ex Steud., is reported. Transgenic plants containing a gus0nptll fusion driven by a double CaMV35S promoter were obtained by microprojectile bombardment of the highly chlorophyllous embryogenic cell line 'TIANSJ98'. Transformed B. gracilis cell lines resisted a lethal concentration of 160 mg/l of kanamycin for at least 8 months. Chlorophyll development and growth rate were used as useful criteria for discriminating transformed from non-transformed clones. Stable integration of the transgene in the blue grama genome was demonstrated by PCR and Southern-hybridization analysis. Expression of the NPTll protein in transgenic plants grown under greenhouse conditions was confirmed indirectly by spraying kanamycin (150-250 mg/l) on plant foliage, and directly by ELISA immunological tests. Control plants sprayed with kanamycin showed foliar necrosis and reduced growth (tillering) compared to plants containing the transgene. NPTll was found in transgenic plants in levels ranging between 12.6 and 29.6 ng/mg FW of cells, as determined by ELISA reactions.     

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein

A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L^-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O₂ in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L^-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.     

Lipid indicators of the diet of the sympagic amphipod Gammarus wilkitzkii in the Marginal Ice Zone and in open waters of Svalbard (Arctic)

Triacylglycerols were the major lipid and wax esters a minor lipid in the Arctic autochthonous, sympagic amphipod, Gammarus wilkitzkii, from less than 1 year old to 3 years old in the Marginal Ice Zone around Svalbard. The fatty acids of the triacylglycerols, especially in young G. wilkitzkii, were mainly characteristic of diatoms and, to a lesser extent, flagellates. Small amounts of 20:1n-9 and 22:1n-11 fatty acids characteristic of calanoid copepods were also present in the triacylglycerols in young G. wilkitzkii from the Marginal Ice Zone and the amounts of both of these fatty acids increased in the triacylglycerols as the animals matured. G. wilkitzkii in open waters in Kongsfjord had minor amounts of triacylglycerols rich in 20:1n-9 and 22:1n-11 and major amounts of wax esters characteristic of calanoid copepods. We conclude that young G. wilkitzkii in the Marginal Ice Zone feed predominantly on ice algae and that they consume increasing amounts of calanoid copepods as they mature, albeit with ice algae remaining a prominent component of their diet. In open waters, young G. wilkitzkii consume mainly calanoid copepods.     

Multi-step feeding systems for lactic acid production by simultaneous saccharification and fermentation of processed wood

Eucalyptus globulus wood samples were delignified in acetic acid media and swelled with NaOH solutions in a further stage. Solid residues from treatments were used as substrates for lactic acid production by Simultaneous Saccharification and Fermentation (SSF) in media containing Trichoderma reesei cellulases and Lactobacillus delbrueckii cells. The improvements in the overall process derived from adding fresh enzymes and/or substrate during the SSF process were assessed. In order to obtain comparative data on the efficiency of substrate utilization, enzymatic hydrolysis runs (in absence of microorganisms) were also carried out. Lactic acid concentrations in the range 48-62 g/l were obtained in SSF experiments. The solid residues after SSF (made up of microbial biomass and the non-hydrolyzed fraction of substrate) were characterized for measuring their potential as feed additives.     

Purification of lysozyme by affinity-based reversed micellar two-phase extraction

A dye-affinity reversed micellar system was used for lysozyme purification from a crude solution of chicken egg white. The dye-affinity reversed micelles consisted of Cibacron Blue F-3GA (CB; 0.1 mM) modified lecithin (50 g/l) in n-hexane. Starting with a crude egg white solution containing lysozyme of 0.0381 mg/mg protein, lysozyme purity was increased by 16 to 20 times, reached 0.62 to 0.76 mg/mg protein. The affinity micellar system was recycled and used three times. Addition of polyoxyethylene (20) sorbitan trioleate (Tween 85) as a cosurfactant could increase the capacity of the affinity-based reversed micelles. A lysozyme recovery yield of over 70% was obtained at a forward aqueous phase pH of 9.16 using the reversed micelles additionally containing 20 g/l of Tween 85.     

Selective extraction using preferential transport through adsorptive membranes

Selective extraction of a protein from a mixture can be accomplished using an adsorptive membrane and low displacement recuperative parametric pumping. Low displacement recuperative parametric pumping can lead to the preferential transport of an adsorbing solute and the rejection of nonadsorbing solutes by the adsorptive membrane. Using a protein mixture consisting of lysozyme and myoglobin, we have found the conditions under which lysozyme is preferentially transported through an ion-exchange membrane cartridge while myoglobin is rejected by the membrane. Trends observed when parameters such as the desorbent concentration, feed concentration, and flow rate are varied agree with the predictions of a mathematical model. Comparison with facilitated diffusion shows that preferential transport can lead to higher solute fluxes, albeit at lower selectivity. Additionally, preferential transport can be used to transport a solute up a concentration gradient and to selectively extract a solute from a feed that contains suspended solids. (c) 1996 John Wiley & Sons, Inc.     

Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose

Citric acid production by Aspergillus niger NCIM 548 and Candida lipolytica NCIM 3472 has been studied in shake culture using glucose and molasses as carbon sources. Methanol addition (3% v/v) at 40 h of fermentation enhanced the production of citric acid by Aspergillus niger whereas a reduction in citric acid production by Candida lipolytica was observed with addition of methanol. Maximum citric acid concentration of 12 kg/m³ was obtained with Aspergillus niger using molasses in the presence of methanol, while maximum citric acid concentration of 8.4 kg/m³ was obtained with Candida lipolytica using glucose without methanol. It appears that product formation by Aspergillus niger is either non-growth associated or partially growth associated depending on the substrate. Methanol addition changes the nature of product formation in case of Candida lipolytica.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Zooplankton life under the perennial Antarctic Sea ice

Zooplankton was collected during the drift on the USA-Russia Ice Station Weddell 1 in the western Weddell Sea. Stratified tows up to 1,000 m depths were performed under perennial ice at a distance of ca. 300 km from the marginal ice zone from March to May 1992. Seasonal abundance, developmental stage composition and vertical distribution in populations of large dominant calanoid copepod species were studied. The abundance of Calanoides acutus changed inconspicuously and averaged 1,260 ind. m^-2; its developmental stage composition was characterised by the dominance of copepodites V (CV) and hardly changed in the course of observations. Calanus propinquus was at most stations in fairly low abundance and was represented by CV and adults; at the latest stations, it was not found. Rhincalanus gigas was also scarce; III-IV stages were present, but the first of them only at the southernmost stations. In contrast to this species, in the relatively abundant population of Metridia gerlachei (1,370 ind. m^-2 on average), a new generation was observed. There was a clear deepening of the median depth of occurrence of the above three species from March to early May, while in late May a shifting to upper layers was observed which remained unexplained. The data obtained were compared to the data collected in the ice-open eastern part of the Weddell Sea in the corresponding season. It can be seen that the differences in populations dynamics of the four species under the perennial ice depends on their trophic mode, i.e. capability to store lipid reserves in summer and/or to feed in winter, and with food spectra.