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The aim of this study was to distinguish effects due to diet composition from those triggered by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system (RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined. Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal grain, 32% hay) diets were more digestible (p < 0.05) than high forage (10% cereal grain, 78% hay) diets, resulting in a greater (p < 0.05) formation of most fermentation end-products and microbial protein in the rumen. However, there were no significant (p > 0.05) differences between diets in methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of oleic acid, and relative proportion of conjugated linoleic acid were increased (p < 0.05) with high forage diets. A decline in rumen pH from 6.8 to 6.4 decreased (p < 0.05) feed digestibility, protein degradability, and the daily outputs of some fermentation end-products (gas, VFA, acetate, ammonia) but had no effect (p > 0.05) on the synthesis of microbial protein, and on the output of methane, propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid relative proportions were observed within this pH range. The overall multivariate analysis revealed a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined pH effects on ruminal fermentation and microbial communities. [ABSTRACT FROM AUTHOR]

The aim of this study was to distinguish effects due to diet composition from those triggered by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system (RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined. Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal grain, 32% hay) diets were more digestible (p < 0.05) than high forage (10% cereal grain, 78% hay) diets, resulting in a greater (p < 0.05) formation of most fermentation end-products and microbial protein in the rumen. However, there were no significant (p > 0.05) differences between diets in methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of oleic acid, and relative proportion of conjugated linoleic acid were increased (p < 0.05) with high forage diets. A decline in rumen pH from 6.8 to 6.4 decreased (p < 0.05) feed digestibility, protein degradability, and the daily outputs of some fermentation end-products (gas, VFA, acetate, ammonia) but had no effect (p > 0.05) on the synthesis of microbial protein, and on the output of methane, propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid relative proportions were observed within this pH range. The overall multivariate analysis revealed a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined pH effects on ruminal fermentation and microbial communities.

Maintaining steady-state, aerobic cultures of yeast in a bioreactor depends on the configuration of the bioreactor system as well as the growth medium used. In this paper, we compare several conventional aeration methods with newer filter methods using a novel optical sensor array to monitor dissolved oxygen, pH, and biomass. With conventional methods, only a continuously stirred tank reactor configuration gave high aeration rates for cultures in yeast extract peptone dextrose (YPD) medium. For filters technologies, only a polydimethylsiloxan filter provided sufficient aeration of yeast cultures. Further, using the polydimethylsiloxan filter, the YPD medium gave inferior oxygenation rates of yeast compared to superior results with Synthetic Complete medium. It was found that the YPD medium itself, not the yeast cells, interfered with the filter giving the low oxygen transfer rates based on the volumetric transfer coefficient (KLa). The results are discussed for implications of miniaturized bioreactors in low-gravity environments. [ABSTRACT FROM AUTHOR]

Large‐scale copepod production is limited by high‐complexity production of microalgae with high contents of polyunsaturated fatty acids (PUFA). However, the tropical A. royi and P. annandalei show great promise of being cultivable on diets with less PUFA content while maintaining both population density and adequate fatty acid (FA) profiles. This ability was investigated through analysis of population density, development stage composition and FA compositions when fed diets of varying FA profiles: the microalgae Dunaliella tertiolecta and Rhodomonas salina, and baker's yeast, Saccharomyces cerevisiae. For A. royi, the population density (>1,600 L−1) did not differ significantly when fed these food items, nor did the relative content of the essential FA, docosahexaenoic acid (C22:6n‐3, DHA), >28% of total FA (p <.05). P. annandalei did not reproduce when fed S. cerevisiae, but population density (>300 L−1) and DHA content (30%–60% DHA of total FA) did not differ significantly when fed D. tertiolecta and R. salina (p <.05). Eicosapentaenoic acid (C20:5n‐3, EPA) content of both copepods differed dependent on the diet, but the resulting DHA:EPA ratio were >2, the optimal for live feed for fish larvae. These findings demonstrate possibilities of utilizing A. royi and P. annandalei in intensive aquaculture, exploiting cheaper diet alternatives, and possibly reducing total production cost and complexity. [ABSTRACT FROM AUTHOR]

In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freezedrying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D=0.1 h-1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rates to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate, and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10% of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D=0.1 h-1 were less robust after freeze-drying and had lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells. [ABSTRACT FROM AUTHOR]

Maintaining steady-state, aerobic cultures of yeast in a bioreactor depends on the configuration of the bioreactor system as well as the growth medium used. In this paper, we compare several conventional aeration methods with newer filter methods using a novel optical sensor array to monitor dissolved oxygen, pH, and biomass. With conventional methods, only a continuously stirred tank reactor configuration gave high aeration rates for cultures in yeast extract peptone dextrose (YPD) medium. For filters technologies, only a polydimethylsiloxan filter provided sufficient aeration of yeast cultures. Further, using the polydimethylsiloxan filter, the YPD medium gave inferior oxygenation rates of yeast compared to superior results with Synthetic Complete medium. It was found that the YPD medium itself, not the yeast cells, interfered with the filter giving the low oxygen transfer rates based on the volumetric transfer coefficient (KLa). The results are discussed for implications of miniaturized bioreactors in low-gravity environments.

Notwithstanding the fundamental role that environmental microbes play for ecosystem functioning, data on how microbes react to disturbances are still scarce, and most factors that confer stability to microbial communities are unknown. In this context, antibiotic discharge into the environment is considered a worldwide threat for ecosystems with potential risks to human health. We therefore tested resilience of microbial communities challenged by the presence of an antibiotic. In a continuous culture experiment, we compared the abundance, composition and diversity of microbial communities undisturbed or disturbed by the constant addiction of tetracycline in low (10 µg/L) or intermediate (100 µg/L) concentration (press disturbance). Further, the bacterial communities in the three treatments had to face the sudden pulse disturbance of adding an allochthonous bacterium (Escherichia coli). Tetracycline, even at low concentrations, affected microbial communities by changing their phylogenetic composition and causing cell aggregation. This, however, did not coincide with a reduced microbial diversity, but was mainly caused by a shift in dominance of specific bacterial families. Moreover, the less disturbed community (10 µg/L tetracycline) was sometimes more similar to the control and sometimes more similar to heavily disturbed community (100 µg/L tetracycline). All in all, we could not see a pattern where the communities disturbed with antibiotics were less resilient to a second disturbance introducing E. coli, but they seemed to be able to buffer the input of the allochthonous strain in a similar manner as the control. [ABSTRACT FROM AUTHOR]

The increasing water crisis makes fresh water a valuable resource, which must be used wisely. However, with growing population and inefficient waste treatment systems, the amount of wastewater dispelled in rivers is increasing abominably. Utilizing this freely available waste-water along with biodiesel industry waste- crude glycerol for bio-hydrogen production is being reported here. The bacterial cultures of Bacillus thuringiensis strain EGU45 and Bacillus amyloliquefaciens strain CD16 produced2.4–3.0 L H2/day/L feed during a 60 days continuous culture system at hydraulic retention time of 2 days. An average H2 yield of 100–120 L/L CG was reported by the two strains. Recycling of the effluent by up to 25% resulted in up to 94% H2 production compared to control. [ABSTRACT FROM AUTHOR]

In the Southern Ocean, natural iron fertilization in the wake of islands leads to annually occurring spring phytoplankton blooms associated with enhanced heterotrophic activity through the release of labile dissolved organic matter (DOM). The aim of this study was to investigate experimentally how diatom-derived DOM affects the composition of Southern Ocean winter water bacterial communities and to identify the most responsive taxa. A bacterial community collected in the naturally iron-fertilized region off Kerguelen Island (KEOPS2 October-November 2011) was grown onboard in continuous cultures, on winter water alone or amended with diatom-derived DOM supplied at identical DOC concentrations. 454 sequencing of 16S amplicons revealed that the two DOM sources sustained strikingly different bacterial communities, with higher relative abundances of Sulfitobacter, Colwellia and Methylophaga operational taxonomic units (OTUs) and lower relative abundances of Polaribacter, Marinobacter, NAC11-7 and SAR11 OTUs in diatom-DOM compared to winter water conditions. Using a modeling approach, we obtained growth rates for phylogenetically diverse taxa varying between 0.12 and 0.49 d-1 under carbon-limited conditions. Our results identify diatom DOM as a key factor shaping Southern Ocean winter water bacterial communities and suggest a role for niche partitioning and microbial interactions in organic matter utilization. [ABSTRACT FROM AUTHOR]

For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness. [ABSTRACT FROM AUTHOR]

Continuous cultures of the marine diatom Thalassiosira pseudonana were grown at irradiances between 6 and 47 µmol m −2 s −1 in order to evaluate the effect of light on hydrogen isotope fractionation in lipids. δ 2 H values increased with irradiance in phytol by 1.1‰ (µmol m −2 s −1 ) −1 and by 0.3‰ (µmol m −2 s −1 ) −1 in the C14:0 fatty acid, but decreased by 0.8‰ (µmol m −2 s −1 ) −1 in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol (C 28 Δ 5,24(28) ). The anticorrelation between δ 2 H values in C 28 Δ 5,24(28) and irradiance is attributed to enhanced sterol precursor synthesis via the plastidic methylerythritol phosphate (MEP) pathway at high irradiance, relative to the cytosolic mevalonic acid (MVA) pathway, and the supposition that MEP precursors are 2 H-depleted compared to MVA precursors because they incorporate a greater proportion of hydrogen from photosynthetically produced NADPH. Increasing δ 2 H values of phytol and C14:0 with irradiance is attributed to a greater proportion of pyruvate, the last common precursor to both lipids, being sourced from glycolysis in the mitochondria and cytosol, where enhanced incorporation of metabolic NADPH and further hydrogen exchange with cell water can enrich pyruvate with 2 H relative to pyruvate from the chloroplast. Irrespective of the biosynthetic mechanisms responsible for the 2 H/ 1 H fractionation response to light, the high sensitivity of lipid δ 2 H values in T. pseudonana continuous cultures would result in −30‰ to +40‰ variations in δ 2 H over a 40 µmol m −2 s −1 range in sub-saturating irradiance if expressed in the environment, depending on the lipid. [ABSTRACT FROM AUTHOR]

Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39°C and exposed to heat shock at 45°C, followed by prolonged heat stress at 45°C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism. [ABSTRACT FROM AUTHOR]

This study was designed to advance understanding of phosphorus regulation of Microcystis aeruginosa growth, phosphorus uptake and storage in changing phosphorus (P) conditions as would occur in lakes. We hypothesized that Microcystis growth and nutrient uptake would fit classic models by Monod, Droop, and Michaelis-Menten in these changing conditions. Microcystis grown in luxury nutrient concentrations was transferred to treatments with phosphorus concentrations ranging from 0–256 μg P∙L-1 and luxury nitrogen. Dissolved phosphorus concentration, cell phosphorus quota, P uptake rate and cell densities were measured at day 3 and 6. Results showed little relationship to predicted models. Microcystis growth was asymptotically related to P treatment from day 0–3, fitting Monod model well, but negatively related to P treatment and cell quota from day 3–6. From day 0–3, cell quota was negatively related to P treatments at <2 μg∙L-1, but increased slightly at higher P. Cell quota decreased greatly in low P treatments from day 3–6, which may have enabled high growths in low P treatments. P uptake was positively and linearly related to P treatment during both periods. Negative uptake rates and increases in measured culture phosphorus concentrations to 5 μg∙L-1 in the lowest P treatments indicated P leaked from cells into culture medium. This leakage during early stages of the experiment may have been sufficient to stimulate metabolism and use of intracellular P stores in low P treatments for rapid growth. Our study shows P regulation of Microcystis growth can be complex as a result of changing P concentrations, and this complexity may be important for modeling Microcystis for nutrient and ecosystem management. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of increasing dietary crude protein (CP) levels and also comparing the effects of static versus oscillating dietary CP on ruminal nutrient digestibility, ruminal fermentation, nitrogen (N) metabolism, and microbial efficiency in beef cattle diets using a dual-flow continuous culture system. Eight fermenters (1,223 ± 21 mL) were used in a replicated 4 x 4 Latin square design with periods lasting 12 d each (8 d for adaptation and 4 d for sampling). Dietary treatments were: 1) 10% CP, 2) 12% CP, 3) 14% CP, and 4) 10 and 14% CP diets oscillating at 48-h intervals. Experimental diets consisted of 50% orchard hay and 50% concentrate. Fermenters were fed 72 g/d and solid and liquid dilution rates were adjusted to 5.5 and 11%/h, respectively. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Apparent and true ruminal digestibilities of dry matter and organic matter were not affected (P > 0.05) by increasing dietary CP, nor by oscillating dietary CP. Total volatile fatty acids concentration and molar proportions of acetate, propionate, butyrate, valerate, iso-butyrate and iso-valerate were not affected (P > 0.05) by increasing or oscillating dietary CP. Ruminal NH3-N concentration increased linearly (P < 0.01) in response to increasing dietary CP. Total N, non-ammonia N, and rumen undegraded protein flows did not differ among treatments or between oscillating dietary CP and static 12% CP. Microbial N and NH3-N flows and microbial efficiency did not differ when comparing oscillating versus static CP (P > 0.05). However, there was a quadratic effect (P < 0.05) for these variables when dietary CP was increased. These results indicate that either ruminal microorganisms do not respond to oscillating CP levels or are capable of coping with 48-h periods of undernourishment. [ABSTRACT FROM AUTHOR]

Depending on the environmental conditions, cells adapt their metabolism and specific growth rate. Rearrangements occur on many different levels such as macromolecular composition, gene and protein expression, morphology and metabolic flux patterns. As the interplay of these processes also determines the output of a recombinant protein producing system, having control over specific growth rate of the culture is advantageous. Continuous culture methods were developed to grow cells in a constant environment and have been used for decades to study basic microbial physiology in a controlled and reproducible manner. Our review summarizes the uses of continuous cultures in cell physiology studies and process development, with a focus on recombinant protein-producing microorganisms.

Bacillus licheniformis ATCC 21415 cells were immobilized on different carriers using different methods of immobilization including physical adsorption, covalent binding, ionic binding and entrapment. The immobilized cells were prepared by covalent binding on wool (as a new carrier) through 1% glutaraldehyde had the highest enzyme activity (9.0 U/mL) with the highest specific productivity (6.17 U/g wet cells/h). Alkaline protease production and the stability of biocatalyst were investigated in both free and immobilized cells. The results showed that the immobilized cells were more efficient for enzyme production by repeated batch fermentation (5 cycles, 480 h) with 57% residual activity whereas the free cells retained 35% after 2 cycles. In continuous production the highest enzyme activity (9.9 U/mL) was obtained at a dilution rate of 0.1/h while the highest enzyme yield (763.6 U/h) and the highest reactor productivity (3.32 U/mL/h) were attained at a dilution rate of 0.4/h. Packed-bed bioreactor was a successful method for continuous production of alkaline protease for a long time (168 h) with 53% relative activity. The bioreactor affected the highest specific productivity (118.2 U/g wet cells/h) which was 12-24 times higher than other systems of enzyme production.

The hydrogen isotopic ( 2 H/ 1 H) composition of lipids in microalgae is significantly depleted relative to extracellular water. While a variety of growth conditions influence the magnitude of 2 H-depletion, the effect of salinity is of particular interest due to the paleohydrological applications of lipid 2 H/ 1 H. In previous studies, lipid–water 2 H/ 1 H fractionation was shown to decrease as salinity increased, a response largely independent of lipid type, species, or setting. The mechanism responsible for this response remains uncertain, primarily because salinity is rarely isolated as the sole variable in laboratory cultivation experiments investigating hydrogen isotope systematics in microalgae. Here we report the lipid–water 2 H/ 1 H fractionation response to salinity in nutrient-replete continuous cultures of the centric diatom Thalassiosira pseudonana . In six cultures with the same growth rate at salinities between 14–40 ppt, lipid–water 2 H/ 1 H fractionation decreased linearly as salinity increased by 1.3‰/ppt in fatty acids (C 14:0 , C 16:0 , C 16:1 ) and by 1.0‰/ppt in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol. A constant growth rate between cultures reveals that the fractionation response to salinity is independent of growth rate. Sensitivity tests using a simple hydrogen flux model indicated that at high salinity a greater proportion of metabolic NAD(P)H in lipids at the expense of photosynthetic NADPH can cause 2 H-enrichment. Additionally, increased exudate release or decreased hydrogen transport can enrich both lipids and cell–water in 2 H. The 1.0–1.3‰/ppt increase in lipid–water 2 H/ 1 H fractionation observed in T. pseudonana is within the 0.8–2‰/ppt range observed in field studies and culture studies, supporting the application of algal lipid 2 H/ 1 H as a paleosalinity proxy. [ABSTRACT FROM AUTHOR]

Salinity and temperature dictate the buoyancy of seawater, and by extension, ocean circulation and heat transport. Yet there remain few widely applicable proxies for salinity with the precision necessary to infer all but the largest hydrographic variations in the past. In the last decade the hydrogen isotope composition ( 2 H/ 1 H or δ 2 H) of microalgal lipids has been shown to increase systematically with salinity, providing a foundation for its use as a paleosalinity proxy. Culture and field studies have indicated a wide range of sensitivities for this response, ranging from about 0.6–3.3‰ ppt −1 depending on the lipid, location and/or culturing conditions. Lacking in these studies has been the controlled conditions necessary to isolate the response to salinity while keeping all other growth parameters constant. Here we show that the hydrogen isotope composition of lipids in the marine coccolithophorid Emiliania huxleyi grown in chemostats increased by 1.6 ± 0.3‰ ppt −1 ( p < 0.05) in eight individual alkenones and by 2.0 ± 0.1‰ ppt −1 ( p < 0.05) in three individual fatty acids over the salinity range 20–42 ppt. Hydrogen isotope ratios of phytol and the sterol 24-methyl-cholest-5,22-dien-3β-ol (brassicasterol) also increased with salinity but correlations were weaker than for the acetogenic lipids. For eight individual alkenones, linear regression analyses of the fractionation factors on salinity yielded slopes of 1.2–2.2‰ ppt −1 . This sensitivity of δ 2 H alkenone to salinity is 45–71% of that previously reported for E. huxleyi , which can be attributed to the fact that previous experiments were performed with batch cultures in which growth rates and other parameters differed between salinity treatments. The underlying cause of this response to salinity remains unknown, but may result from changes in (1) the proportion of lipid hydrogen derived from NADPH versus water, (2) the proportion of lipid hydrogen derived from NADPH from Photosystem I versus the oxidative pentose phosphate pathway (and other metabolic sources), or (3) the δ 2 H value of intracellular water. [ABSTRACT FROM AUTHOR]

Excessive levels of starch in diets for lactating dairy cattle is a known risk factor for milk fat depression, but little is known about how this risk is affected by differences in rates of starch degradability (Kd) in the rumen. The objective of this study was to compare accumulation of biohydrogenation intermediates causing milk fat depression, including conjugated linoleic acid (CLA), when corn with low or high Kd were fed to continuous cultures. Diets contained (dry matter basis) 50% forage (alfalfa pellets and grass hay) and 50% concentrate, with either no added fat (LF) or 3.3% added soybean oil (HF). Within both the LF and HF diets, 3 starch degradability treatments were obtained by varying the ratio of processed (heat and pressure treatments) and unprocessed corn sources, giving a total of 6 dietary treatments. Each diet was fed to dual-flow continuous fermenters 3 times a day at 0800, 1600, and 2400 h. Diets were fed for four 10-d periods, with 7 d for adaptation and 3 d for sample collection. Orthogonal contrasts were used in the GLIMMIX procedure of SAS to test the effects of fat, starch degradability, and their interaction. Acetate and acetate:propionate were lower for HF than for LF but daily production of trans-10 18:1 and trans-10,cis-12 CLA were higher for HF than for LF. Increasing starch Kd from low to high increased culture pH, acetate, and valerate but decreased butyrate and isobutyrate. Changes in biohydrogenation intermediates (expressed as % of total isomers) from low to high starch Kd included reductions in trans-11 18:1 and cis-9,trans-11 CLA but increases in trans-10 18:1 and trans-10,cis-12 CLA. The results show that increasing the starch Kd in continuous cultures while holding starch level constant causes elevation of biohydrogenation intermediates linked to milk fat depression. [ABSTRACT FROM AUTHOR]

The growth characteristics, lipid accumulation and composition during the life cycle of a newly isolated strain of Debaryomyces etchellsii were studied under nitrogen limiting conditions. This yeast, grown in batch flask or bioreactor cultures, reproduced asexually by buds when nitrogen was available in the growth medium, or sexually by ascospores after nitrogen exhaustion, producing more than 7 g L −1 biomass. During ascosporogenesis, an important increase in the cellular lipid content in dry cell mass occurred, i.e. from a mass fraction of 11.9% in the vegetative phase to 22.4%, in the ascosporogenic phase. During transition of D . etchellsii from batch to continuous cultures using dilution rates 0.026 and 0.019 h −1 , a shift from sexual to asexual reproduction was observed. At 0.019 h −1 , few pseudomycelia were also formed. The yeast synthesized lipids containing long chain fatty acids (mainly C16 and C18). Budded cells at steady-states contained only 8.6–9.3 % of lipids mass fraction per dry cell mass that were composed of oleic and linoleic acids and, to a lesser extent, of palmitic and palmitoleic acids. Neutral lipids were the major fraction represented 61.8–66.1%, of total lipids followed by phospholipids, which was the only fraction in which linoleic acid predominated over oleic acid. [ABSTRACT FROM AUTHOR]

The effect of pH and dilution rate on the production of extracellular metabolites of Lactobacillus salivarius UCO_979 was studied. The experiments were carried out in continuous mode, with chemically defined culture medium at a temperature of 37 °C, 200 rpm agitation and synthetic air flow of 100 ml/min. Ethanol, acetic acid, formic acid, lactic acid and glucose were quantified through HPLC, while exopolysaccharide (EPS) was extracted with ethanol and quantified through the Dubois method. The results showed no linear trends for the specific production of lactic acid, EPS, acetic acid and ethanol, while the specific glucose consumption and ATP production rates showed linear trends. There was a metabolic change of the strain for dilution rates below 0.3 h. The pH had a significant effect on the metabolism of the strain, which was evidenced by a higher specific glucose consumption and increased production of ATP at pH 6 compared with that obtained at pH 7. This work shows not only the metabolic capabilities of L. salivarius UCO_979C, but also shows that it is possible to quantify some molecules associated with its current use as gastrointestinal probiotic, especially regarding the production of organic acids and EPS. [ABSTRACT FROM AUTHOR]

The fate of allochthonous dissolved organic carbon (DOC) in aquatic systems is primarily controlled by the turnover of heterotrophic bacteria. However, the roles that abiotic and biotic factors such as light and DOC release by aquatic primary producers play in the microbial decomposition of allochthonous DOC is not well understood. We therefore tested if light and autochthonous DOC additions would increase allochthonous DOC decomposition rates and change bacterial growth efficiencies and community composition (BCC). We established continuous growth cultures with different inocula of natural bacterial communities and alder leaf leachates (DOC) with and without light exposure before amendment. Furthermore, we incubated DOC together with autochthonous DOC from lysed phytoplankton cultures (DOC). Our results revealed that pretreatments of DOC with light resulted in a doubling of bacterial growth efficiency (BGE), whereas additions of DOC or combined additions of DOC and light had no effect on BGE. The change in BGE was not accompanied by shifts in the phylogenetic structure of the BCC, but BCC was influenced by the DOC source. Our results highlight that a doubling of BGE is not necessarily accompanied by a shift in BCC and that BCC is more strongly affected by resource properties. [ABSTRACT FROM AUTHOR]

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods. [ABSTRACT FROM AUTHOR]

Respiratory epithelial cells play a key role in influenza A virus (IAV) pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV−host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77) IAVs in normal primary human bronchial epithelial (NHBE) and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK) cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7), stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK) inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo. [ABSTRACT FROM AUTHOR]

Biofilms are microbial communities attached to a surface and embedded in a matrix composed of exopolysaccharides and excreted nucleic acids. Bacterial biofilms are responsible for undesirable effects such as disease, prostheses colonization, biofouling, equipment damage, and pipe plugging. Biofilms are also more resilient than free-living cells to regular sterilization methods and therefore it is indispensable to develop better ways to control and remove them. The use of gas discharge plasmas is a good alternative since plasmas contain a mixture of reactive agents well-known for their decontamination potential against free microorganisms. We have previously reported that Pseudomonas aeruginosa biofilms were inactivated after a 1-min plasma exposure. We determined that the adhesiveness and the thickness of Pseudomonas biofilms grown on borosilicate were reduced. We also reported sequential morphological changes and loss of viability upon plasma treatment. However, the studies were carried out in batch cultures. The use of a continuous culture results in a more homogenous environment ensuring reproducible biofilm growth. The aim of this work was to study plasma-mediated inactivation of P. aeruginosa biofilms grown on borosilicate in a continuous culture system. In this paper we show that biofilms grown on glass under continuous culture can be inactivated by using gas discharge plasma. Both biofilm architecture and cell culturabilty are impacted by the plasma treatment. The inactivation kinetics is similar to previously described ones and cells go through sequential changes ranging from minimal modification without loss of viability at short plasma exposure times, to major structure and viability loss at longer exposure times. We report that changes in biofilm structure leading to the loss of culturability and viability are related to a decrease of the biofilm matrix adhesiveness. To our knowledge, there has been no attempt to evaluate the inactivation/sterilization of biofilms grown in a continuous system. [ABSTRACT FROM AUTHOR]

The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH2) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH2 NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH2 NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for in vitro screening of nanomaterials in heighthroughput format. [ABSTRACT FROM AUTHOR]

A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an in vivo-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., Pseudomonas aeruginosa) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-κB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca2+-channels but they exhibited increased intracellular Ca2+ levels in response to ATP. These Ca2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches. [ABSTRACT FROM AUTHOR]

In this paper, robust control problem using μ-synthesis in microbial continuous culture is studied. The dissimilation process of glycerol to 1,3-propanediol cannot avoid the disturbances caused by uncertain factors. Based on the biodynamical model, a control system with the initial glycerol concentration as input control is proposed to simplify the controller design. μ-synthesis method is applied to find a feedback controller to assure both of robust stability and robust performance of the closed-loop system simultaneously. To solve the corresponding structured singular value optimization problem, a converged result is obtained through D-K iteration method. The μ-synthesis system is also compared with the corresponding $$H_\infty$$ system. The simulation results indicate that the μ-controller might be more feasible for the continuous bioprocess controlling. [ABSTRACT FROM AUTHOR]

In this paper, stability analysis and controller design in microbial continuous culture with discrete time delay are studied. The dissimilation process of glycerol to 1,3-propanediol cannot avoid the disturbances caused by time delay. Time delay can limit and degrade the achievable performance of controlled systems, and even induce instability. Based on the biodynamic model, some properties of its solutions are discussed. In addition, we investigate how the time-delay affects the stability of the system. A linear matrix inequalities method is applied to find a feedback controller to ensure the stability of the closed-loop system. The simulation results indicate that this controller might be feasible for continuous bioprocess control. [ABSTRACT FROM AUTHOR]

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. [ABSTRACT FROM AUTHOR]

The reduction of polyphenols content in olive mill wastewater (OMW) is a major issue in olive oil manufacturing. Although researchers have pointed out the potential of white-rot fungus in dephenolizing OMW, the results available in the literature mainly concern pretreated (sterilized) OMW. This paper deals with the reduction of polyphenols content in untreated OMW by means of a white-rot fungus, Pleurotus ostreatus. Dephenolization was performed both in an airlift bioreactor and in aerated flasks . The process was carried out under controlled non-sterile conditions, with different operating configurations (batch, continuous, biomass recycling) representative of potential industrial operations. Total organic carbon, polyphenols concentration, phenol oxidase activity, dissolved oxygen concentration, oxygen consumption rate, and pH were measured during every run. Tests were carried out with or without added nutrients (potato starch and potato dextrose) and laccases inducers (i.e., CuSO). OMW endogenous microorganisms were competing with P. ostreatus for oxygen during simultaneous fermentation. Dephenolization of raw OMW by P. ostreatus under single batch was as large as 70%. Dephenolization was still extensive even when biomass was recycled up to six times. OMW pre-aeration had to be provided under continuous operation to avoid oxygen consumption by endogenous microorganisms that might spoil the process. The role of laccases in the dephenolization process has been discussed. Dephenolization under batch conditions with biomass recycling and added nutrients proved to be the most effective configuration for OMW polyphenols reduction in industrial plants (42-68% for five cycles). [ABSTRACT FROM AUTHOR]

Considering the inhibition of 3-HPA. to the growth of cells and the mode of transmemberane transport between substrate and product, a novelty mathematical model is established to describe the microbial continuous cultures better. Taking the mean minimal error between calculated values and the experimental data of steady state as the performance index, a parameter identification model involving multiple dynamic systems is presented. The identifiability of the model is also proved. An improved particle swarm optimization (PSO) algorithm is constructed to solve the parameter identification model. Numerical results show that the established model can describe the microbial continuous cultures process better. [ABSTRACT FROM AUTHOR]

Although bacteriophage have been reported to be the most abundant organisms on earth, little is known about their contribution to the ecology of natural communities of their host bacteria. Most importantly, what role do these viral parasitoids play in regulating the densities of bacterial populations? To address this question, we use experimental communities of Vibrio cholerae and its phage in continuous culture, and we use mathematical models to explore the population dynamic and evolutionary conditions under which phage, rather than resources, will limit the densities of these bacteria. The results of our experiments indicate that single species of bacterial viruses cannot maintain the density of V. cholerae populations at levels much lower than that anticipated on the basis of resources alone. On the other hand, as few as two species of phage can maintain these bacteria at densities more than two orders of magnitude lower than the densities of the corresponding phage-free controls for extensive periods. Using mathematical models and short-term experiments, we explore the population dynamic processes responsible for these results. We discuss the implications of this experimental and theoretical study for the population and evolutionary dynamics of natural populations of bacteria and phage. [ABSTRACT FROM AUTHOR]

Abstract: It has been reported that some condensed tannin (CT) containing forage legumes may reduce ruminal CH4 emissions as a result of direct and/or indirect inhibition of methanogens. Dual flow continuous fermentors (working volume of 700ml) were used to determine effects of CT containing legume hays in dairy total mixed rations (TMR) on ruminal fermentation, with an emphasis on CH4 and ammonia N production. Fermentors were inoculated with filtered rumen contents and allowed to adapt to experimental diets for 6d, followed by 3d of sampling and data collection. All dietary treatments were formulated for lactating dairy cows in early lactation. Five TMR were evaluated being: (1) alfalfa (AFT), (2) cicer milkvetch (CMVT), (3) Norcen birdsfoot trefoil (NBFTT), (4) Oberhaunstadter birdsfoot trefoil (OBFTT) and (5) sainfoin (SFT). The experiment was a 5×5 Latin square design. Compared to AFT, culture pH was not influenced by most forages, but was increased (P<0.01) by feeding SFT. Compared to AFT, the molar proportion of propionate increased (P=0.03) with CMVT, but decreased (P=0.05) with SFT. Feeding CMVT decreased (P<0.01) the molar proportion of butyrate relative to AFT. Acetate to propionate ratio decreased with CMVT (P<0.01) and OBFTT (1.65; P=0.01), but increased with SFT (P<0.01) compared to AFT. Apparent digestibility of dry matter or neutral detergent fiber (aNDF) was not influenced by forage source. Compared to AFT, CH4 production decreased with other treatments (P<0.01), whereas CH4 production (expressed as mM/g aNDF digested) was only reduced (P<0.05) by CMVT and OBFTT. NH3–N concentration was lower (P<0.01) with CMVT and SFT, but not affected by NBFTT or OBFTT compared to AFT. Legume forages eliciting reductions in CH4 and NH3–N production may offer advantages in lactating dairy diets to improve nutrient utilization and environmental sustainability of dairy cattle production. This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson. [Copyright &y& Elsevier]

Abstract: During lactic acid fermentation, seed culture is usually carried out without pH control, while culture is carried out at pH controlled at the optimal value to overcome inhibitory effects. The Luedeking–Piret expression was therefore previously modified by introducing additional terms involving the undissociated form of the lactic acid, the main inhibitory species, in case of batch cultures without pH control or involving the residual lactose concentration to account for the carbon substrate limitation, responsible for cessation of production during batch cultures of Lactobacillus helveticus at controlled pH. Both expressions were also merged to deduce a generalized model. Both models, as well as the Luedeking–Piret model, were developed to describe continuous two-stage culture of L. helveticus. By considering the parameter values obtained from the fitting of batch culture data, both modified Luedeking–Piret models showed interesting predictive potential. Indeed, some rather reliable predictive calculated values were recorded in both stages; the residual standard deviations were 0.5 and less than 8.8 for the biomass and the product concentrations at steady-state in the culture stage (second stage). The optimization of the parameters for growth- and non-growth-associated parameters improved the fitting in the culture stage, leading to residual standard deviations below 2.6 for lactic acid concentrations at steady-state. [Copyright &y& Elsevier]

Abstract: The effects of nutrient limitations on hydrogen yields during fermentation by chemostat cultures of Escherchia coli were studied. Four nutrient regimes (limiting nutrients) were chosen; glucose, nitrogen, sulfur and phosphate. Yields with limiting phosphate or sulfate were low, at or below 1mol glucose. Yields with continuous cultures limited for nitrogen decreased with increasing dilution rate , and were lower than that obtained with a static culture . The highest yields (at, or somewhat higher than 2mol glucose) were obtained with cultures limited for glucose. Under most conditions, total hydrogen production and yield were inversely related, with higher production, but lower yields, at increasing dilution rate. A strain, DJT135, mutated in the uptake hydrogenases, in lactate dehydrogenase, and with constitutively active FhlA gave the highest production and greatest to glucose yields. [Copyright &y& Elsevier]

Abstract: In a test of the biotic ligand model (BLM), the uptake and toxicity of silver, in the absence or presence of the inorganic ligand, thiosulphate, were assessed for two freshwater green algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata, using turbidostat continuous cultures. In the initial experiments, run in the absence of thiosulphate, the influent Ag concentration was varied from 0 to 75nM in steps; for each influent concentration, silver uptake was calculated and the algal growth rate was determined. Silver uptake rates at low Ag concentrations were similar for both algae (e.g., 14–19nmolm−2 h−1, for influent Ag+ concentrations of ∼9nM) but at higher exposures uptake by P. subcapitata exceeded that of C. reinhardtii. Despite this higher uptake rate, in the absence of thiosulphate P. subcapitata was not more sensitive to free silver; 50% growth inhibition was reached at influent free Ag+ concentrations of 15±7 and 22±13nM for C. reinhardtii and P. subcapitata, respectively. In the second series of experiments, the free Ag+ concentration was held constant (∼9nM in the influent; 2–3nM in the effluent) while the concentration of the silver thiosulphate complex, AgS2O3 −, was increased from 9 to 90nM in steps. Under such conditions, the BLM would predict that silver uptake and toxicity should remain constant. On the contrary, both silver uptake and silver toxicity increased, indicating that the anionic silver thiosulphate complex enters the algal cells via a membrane-bound sulphate transporter and contributes to uptake and toxicity. However, for both algae there were indications that silver assimilated in this manner was somewhat less toxic to the algal cell than silver that entered via cation transport only. Physiological indicators of stress revealed possible different intracellular targets for these two freshwater algae, proteins and enzymes for C. reinhardtii and the photosynthetic apparatus for P. subcapitata. [Copyright &y& Elsevier]

Abstract: Kinetic modeling of rhIFN-α expression was done in continuous cultures using complex media at dilution rates varying from 0.2 to 0.5h−1. The human IFN-α gene was inserted under the T7 promoter in BL21 (DE3) codon plus (RIL) cells and induced with 1mM IPTG. Post-induction growth and production profiles were monitored over time. Wash out of the cells was observed only at a dilution rate of 0.5h−1, which gave a post-induction specific growth rate (μ) of 0.37h−1. The rate of accumulation of rhIFN-α inside the cells as inclusion bodies was used to calculate the specific product formation rate (q p). q p was found to be strongly correlated with the dilution rates used with the maximum q p being 0.17gg−1 h−1 at the first hour of induction at a dilution rate of 0.5h−1.The decline in q p with time could be related to the build up of inclusion bodies inside the cells which reached a maximum value of 50% of the total protein in the fourth hour post-induction. [Copyright &y& Elsevier]

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1.3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l-1. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l-1, at D=0.05-0.3 h-1, and from 60-70 g l-1, at D=0.05 and 0.1 h-1 · 30 g l-1 of 1,3-propanediol and the highest reported value of productivity, 10.3 g l-1 h-1, was achieved at D = 0.30 h-1 and 60 g l-1 of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l-1 of feed glycerol and a dilution rate higher than 0.10 h-1; moreover, at D = 0.30 h-1 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum. [ABSTRACT FROM AUTHOR]

The purpose of this study was to explore the role of glycogen and trehalose in the ability of Saccharomyces cerevisiae to respond to a sudden rise of the carbon flux. To this end, aerobic glucose-limited continuous cultures were challenged with a sudden increase of the dilution rate from 0.05 to 0.15 h-1. Under this condition, a rapid mobilization of glycogen and trehalose was observed which coincided with a transient burst of budding and a decrease of cell biomass. Experiments carried out with mutants defective in storage carbohydrates indicated a predominant role of glycogen in the adaptation to this perturbation. However, the real importance of trehalose in this response was veiled by the unexpected phenotypes harboured by the tps1 mutant, chosen for its inability to synthesize trehalose. First, the biomass yield of this mutant was 25% lower than that of the isogenic wild-type strain at dilution rate of 0.05 h-1, and this difference was annulled when cultures were run at a higher dilution rate of 0.15 h-1. Second, the tps1 mutant was more effective to sustain the dilution rate shift-up, apparently because it had a faster glycolytic rate and an apparent higher capacity to consume glucose with oxidative phosphorylation than the wild type. Consequently, a tps1gsy1gsy2 mutant was able to adapt to the dilution rate shift-up after a long delay, likely because the detrimental effects from the absence of glycogen was compensated for by the tps1 mutation. Third, a glg1Δglg2Δ strain, defective in glycogen synthesis because of the lack of the glycogen initiation protein, recovered glycogen accumulation upon further deletion of TPS1. This recovery, however, required glycogen synthase. Finally, we demonstrated that the rapid breakdown of reserve carbohydrates triggered by the shift-up is merely due to changes in the concentrations of hexose-6-phosphate and UDPglucose, which are the main metabolic effectors of the rate-limiting enzymes of glycogen and trehalose pathways. [Copyright &y& Elsevier]