Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. (c) 1996 John Wiley & Sons, Inc.     

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.     

The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation

Summary Rhizoferrin is a novel carboxylate-type siderophore which has recently been isolated fromRhizopus microsporus and other fungi of the Mucorales (Zygomycetes). The present investigation shows that a variety of rhizoferrin analogs can be produced by directed fermentation. Thus both the diaminobutane backbone and the citric acid side chains of rhizoferrin have been substituted by diamine and citric acid analogs added to the culture medium. The new ligands as well as their iron complexes have been characterized by physicochemical methods. Conditions of precursor incorporation and implications for the biosynthesis of the new siderophores are discussed.     

The upper cretaceousLacazina limestone in the Basco-Cantabrian and Iberian basins of northern Spain: Cold-water grain associations in warm-water environments

Summary The Upper Santonian to Lower CampanianLacazina Limestone consists of massive, often amalgamated beds of packstones and grainstones which were deposited in a shallow marine environment. The most abundant skeletal components are miliolid foraminifera, echinoderm, bivalve and bryozoan fragments, peloids and sparse red algae. Small, solitary corals only occur sporadically. Hermatypic corals, sponges and green algae are missing. The series which reaches thicknesses between 60 m and 160 m, was sampled at intervals of 0.5 m at five localities. The petrographic features throughout the series are mainly a product of changing depositional energy. The limestones are well cemented. Diagenesis is characterized by a transition from marine phreatic to burial cementation. Syntaxial and blocky calcite cements predominate over early acicular to bladed and microgranular cements. The faunal association within theLacazina Limestone exhibits features typical for temperate water i.e.foramol carbonates. On the other hand, oxygen (δ¹⁸O =-1.7 to −6.3 ‰ PDB) and carbon (δ¹³C to 3.0‰ PDB) isotope values of diagentically unaltered bivalve shells indicate warm surface waters corresponding better to the palaeogeographical situation of theLacazina Limestone. Nutrient-surplus is proposed as a limiting factor preventing the development of reefs and finally ofchlorozoan sediments. In the sense of sequence stratigraphy, theLacazina Limestone is interpreted to contain transgressive and highstand systems tracts. This interpretation fits well into theHaq-Vail-curve. The series shows no visible high-frequency cyclicity in the field. Several cycles were found by means of principle component analysis and spectral analysis. Their relationships to the Milankovitch spectrum are discussed. The ammonite fauna of the unit and of preceding sediments (late Coniacian to early Campanian) is described and some inoceramids are figured. They permit—for the first time—the exact dating of theLacazina Limestone in the Basco-Cantabrian Basin (BCB) and throw light on a prominent faunal change at the Coniacian/Santonian boundary. The Cenomanian to Coniacian ammonite faunas which were dominated by endemic Tethyan pseudoceratitic faunas are replaced by cosmopolitan species dominated by Madagascan elements. This drastic change permits speculations about the installation of a new oceanic current system in the Santonian.     

The influence of cosubstrate and aeration on xylitol formation by recombinantSaccharomyces cerevisiae expressing theXYL1 gene

Xylitol formation by a recombinantSaccharomyces cerevisiae strain containing theXYL1 gene fromPichia stipitis CBS 6054 was investigated under three sets of conditions: (a) with glucose, ethanol, acetate, or glycerol as cosubstrates, (b) with different oxygenation levels, and (c) with different ratios of xylose to cosubstrate. With both glucose and ethanol the conversion yields were close to 1 g xylitol/g consumed xylose. Decreased aeration increased the xylitol yield on the basis of consumed cosubstrate, while the rate of xylitol formation decreased. The xylitol yield based on consumed cosubstrate also increased with increased-xylose:cosubstrate ratios. The transformant utilized the cosubstrate more efficiently than did a reference strain in terms of utilization rate and growth rate, implying that the regeneration of NAD(P)⁺ during xylitol formation by the transformant balanced the intracellular redox potential.     

Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH)

The use of a plate screening technique allowed the direct isolation and quantification of polycylic aromatic hydrocarbon (PAH)-degrading bacteria from different soil sites. Bacteria that were able to grow on anthracene, phenanthrene, fluoranthene or pyrene as a sole carbon source were found with numbers between 10³ and 10⁵ colony-forming units (cfu)/g of soil dry weight, but only in samples that originated from PAH-contaminated sites. No isolates were found that could grow on perylene, triphenylene, benzo(a)pyrene or chrysene as sole carbon source. Bacteria that had been selected on the same PAH substrate showed a related degradation pattern for both other PAH and oil compounds and carbohydrate substrates even if they had been collected at distant soil sites. Based on these findings the isolates could be clustered into four different catabolic and taxonomic similarity groups. Taxonomic determination of representative isolates suggested that nocardioform actinomycetes of the genera Mycobacterium, Rhodococcus and Gordona represented a major part of the soil microflora able to mineralize PAH. Three new isolates able to grow on anthracene, pyrene or fluoranthene as the sole carbon source, respectively, have been isolated and identified (Sphingomonas paucimobilis BA2, Gordona sp. BP9, Mycobacterium sp. VF1). The ubiquitous presence of a potent and versatile mineralizing microflora in PAH-contaminated soils indicated that the microflora is not the limiting factor for the degradation of PAH with up to four rings.     

Fasting endurance and cold resistance without hypothermia in a small predatory bird: the metabolic strategy of Tengmalm's owl,Aegolius funereus

The energetic adaptations of non-breeding Tengmalm's owls (Aegolius funereus) to temperature and fasting were studied during the birds' autumnal irruptions in western Finland. Allometric analysis (including literature data and two larger owl species measured in this study) indicates that the basal metabolic rate of owls is below the mean level of non-passerine birds. However, the basal metabolic rate of the 130-g Tengmalm's owl (1.13 W) is higher than in other owls of similar size. This is probably related to its northern distribution and nomadic life history. Relative to its size, Tengmalm's owl has excellent cold resistance due to effective insulation (lower critical temperature +10°C, minimum conductance 0.19 mW·cm^-2·°C^-1). Radiotelemetric measurements of body temperature showed that the level of body temperature is lower than for birds in general (39.4°C at zero activity) and that the amplitude of the diurnal cycle is also low (0.2–0.6°C). In contrast to many other small birds, Tengmalm's owls do not enter hypothermia during a 5-day fast at thermoneutrality or in cold. Moreover, while the metabolic rate per bird shows the expected mass-dependent decrease, the mass-specific rate decreases only slightly during the fast. In line with this, there was no decrease in the plasma triiodothyronine concentration during the fast in the owl, whereas a dramtic drop was observed in the pigeon and Japanese quail that were used as a reference. Despite this, the owl has an excellent capacity for fasting because of its ability to accumulate extensive fat depots and its low overall metabolic rate. Fasting reduced evaporative water loss to 50% of that in the fed state. Calculations show that the oxygen consumption observed in fasting birds would involve a production of metabolic water barely sufficient to compensate for evaporative water loss. The threat of dehydration may thus set a limit to the decrease in metabolic rate in fasting owls (owls rely totally on water either ingested with food or produced metabolically). We conclude that the metabolic strategy in Tengmalm's owl is largely dictated by an evolutionary pressure for fasting endurance. With the restrictions set by small body size and water economy, this bird has apparently taken these adaptations to an extreme. The constraints that preclude hypothermia, which could increase the capacity for fasting even more, remain unknown.Abbreviations BM body mass - BMR basal metabolic rate - EWL vaporative water loss - MR metabolic rate - T₃ triiodothyronine - T _a ambient temperature - T _b body temperature - VO₂ oxygen consumption     

Growth of rat pancreatic acinar cells quantitated with a monoclonal antibody against the proliferating cell nuclear antigen

The monoclonal antibody PC10 raised against the proliferating cell nuclear antigen (PCNA) was used to study acinar cell replication in the pancreas of rats under different functional conditions. In Western blots, the antibody recognized a single band of 37 kDa in pancreatic homogenates indicating its specificity in this particular species and organ. Three conditions of growth were chosen for immunohistochemical analysis: pancreatic preand postnatal development, pancreatic regeneration after injury, and cholecystokinin-stimulated acinar cell proliferation. The time course of acinar cell replication under each condition was the same as that obtained after tritiated thymidine incorporation with subsequent autoradiography, indicating that the percentage of PCNA-positive cells reflects the pool of cycling cells in the models investigated. However, the absolute number of PCNA-positive cells was two to ten times higher than comparable labeling indices from ³H-thymidine autoradiography. This finding might reflect the half life of PCNA, which exceeds the duration of the S-phase. Thus, PCNA-positive cells not only represent S-phase cells, but also cells that have recently completed the cell cycle.     

Chemical composition of lipopolysaccharides from Legionella bozemanii and Legionella longbeachae

Lipopolysaccharides (LPS) from Legionella bozemanii serogroup 1 and Legionella longbeachae serogroup 1 were subjected to chemical analyses. The lipid A part of both LPSs contained 2,3-dideoxy-2,3-diamino-d-glucose as major constituents and d-glucosamine and glycerol as minor constituents of the sugar backbone structure. Both LPSs exhibited a very complex fatty acid composition. Twenty amide-linked 3-hydroxy fatty acids were detected in LPS of L. longbeachae, whereas seventeen were encountered in LPS of L. bozemanii. Both LPSs contained nine ester-linked nonhydroxy fatty acids and the unique long-chain fatty acids 27-oxo-octacosanoic acid, 29-oxotriacontanoic acid, heptacosane-1,27-dioic acid and nonacosane-1,29-dioic acid. SDS-PAGE showed that L. bozemanii produced smooth-form LPS, whereas L. longbeachae LPS was mainly of the R-type. Composition analyses were in accordance with these electrophoretic patterns. d-Quinovosamine and l-fucosamine constituted 80 mol% of the polysaccharide part of L. bozemanii LPS. Other sugars identified were d-glucosamine, d-mannose, d-glucose, l-rhamnose, d-glycero-d-manno-heptose, l-glycero-d-mannoheptose, 2-keto-3-deoxy-octonic acid and glycerol. The polysaccharide chain from LPS of L. longbeachae appeared to be shorter, but composed of the same sugars except l-fucosamine. Both LPSs contained glycerol phosphate and glucosamine phosphate and L. longbeachae LPS contained in addition glucose phosphate.Abbreviations EI Electron impact - GlcN3N 2,3-Diamino-2,3-dideoxy-d-glucose - HPAEC High pH anion-exchange chromatography - Kdo 2-Keto-3-deoxy-octonic acid - LPS Lipopolysaccharide - PCP Phenol/chloroform/petroleum ether solvent - PED Pulsed electrochemical detection - PS Polysaccharide - TFA Trifluoroacetyl - TMS Trimethylsilyl