High Dilution Rates Research Articles

Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 106/mL, 5 × 106/mL, and 1 × 106/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 106/mL dilution rate, spermatozoa diluted to 1 × 106 sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.

Effect of the Feed Substrate Concentration on the Dynamic Performance of the Bioethanol Fermentation Process Using Zymomonas mobiliz

In this work, a structured verified nonlinear mathematical model for a single continuous fermenter of bioethanol production using Zymomonas mobiliz is developed to investigate the effect of the feed substrate concentration, which is taken as the main bifurcation parameter for performing a bifurcation analysis. The chaotic, oscillatory, and steady-state behaviors of the fermentation system are investigated. It is found that the system is dominated by oscillatory behavior in the medium gravity (MG) region (80.76 < Cso < 213.6), while it is dominated by stable steady state outside this range; however, at high Cso and low dilution rate, the chaotic behavior dominates the system. It is found that the fermentation system accomplishes the maximum averages of substrate conversion and bioethanol yield in the range of 80.76 < Cso < 97 g/L. These results are in agreement with the experimental results in the literature.

Impact of Steam-Dilution on the Flame Shape and Coherent Structures in Swirl-Stabilized Combustors

Humidified gas turbines and steam-injected gas turbines are promising technologies to lower the emissions and increase the efficiency and fuel flexibility of gas turbines. In the current study, the influence of steam-dilution on swirl-stabilized methane and hydrogen-fired flames is experimentally investigated at Reynolds numbers in the range of 22,000 to 32,000. Velocity fields and flame positions were measured using high-speed particle image velocimetry and OH* chemiluminescence. An extension of the quantitative light sheet technique was employed to estimate the temperature fields. The combined results reveal strong changes in the flame position, the velocity field, and the temperature field with increasing rates of steam dilution. In particular, three different flow and flame patterns are encountered: At dry conditions, a V-shaped flame stabilizes in a broad inner recirculation zone with low local turbulent kinetic energy; at moderate steam content, the flame changes into a trumpet-like shape; and at very high rates of steam-dilution, the flame detaches and shows an annular shape. The associated coherent flow structures are extracted from the particle image velocimetry data employing proper orthogonal decomposition. The isothermal flow is dominated by a helical instability arising near the combustor inlet. This structure is completely suppressed for the dry flame and reappears for the heavily steam-diluted detached flame with a similar shape and frequency as for the isothermal case. The flow field of the trumpet-like flame at intermediate to high steam dilution rates features a helical instability of lower frequency that is located further downstream than in the isothermal and very wet case. A conceptional explanation is presented that relates the suppression of the helical instability to the specific encountered temperature fields and flame shapes.

Fermentation of lignocellulosic hydrolyzate using a submerged membrane bioreactor at high dilution rates.

A submerged membrane bioreactor (sMBR) was developed to ferment toxic lignocellulosic hydrolyzate to ethanol. The sMBR achieved high cell density of Saccharomyces cerevisiae during continuous cultivation of the hydrolyzate by completely retaining all yeast cells inside the sMBR. The performance of the sMBR was evaluated based on the ethanol yield and productivity at the dilution rates 0.2, 0.4, 0.6, and 0.8h(-1) with the increase of dilution rate. Results show that the yeast in the sMBR was able to ferment the wood hydrolyzate even at high dilution rates, attaining a maximum volumetric ethanol productivity of 7.94 ± 0.10 g L(-1)h(-1) at a dilution rate of 0.8h(-1). Ethanol yields were stable at 0.44 ± 0.02 g g(-1) during all the tested dilution rates, and the ethanol productivity increased from 2.16 ± 0.15 to 7.94 ± 0.10 g L(-1)h(-1). The developed sMBR systems running at high yeast density demonstrate a potential for a rapid and productive ethanol production from wood hydrolyzate.

Simulation of the rate of transfer of antibiotic resistance between Escherichia coli strains cultured under well controlled environmental conditions

It was demonstrated that the tetracycline resistance plasmid in Escherichia coli resembling K-12 23:06 containing the E. coli plasmid DM0133 could be transferred to tetracycline sensitive E. coli K-12 MG1655 YFP. The sensitive recipient strain has a slight metabolic advantage in continuous fermentation in absence of tetracycline pressure and as a result the numbers of the resistant recipient strain increase during fermentation. In presence of tetracycline pressure the sensitive strain is eliminated, but when it acquires tetracycline resistance the strain has still the same metabolic advantage as its sensitive parent strain in absence of tetracycline. Here a model will be shown that could explain the rate of transformation of a sensitive into a resistant recipient strain and its subsequent growth during continuous fermentation. According to the model the probability of formation of mutants would be much higher at the dilution rate of 0.09 compared to 0.28, whereas the growth of mutants would be much faster at high dilution rate. The growth model shows how the recipient mutants and the donor cells behave in relation to the dilution rate and the number of mutants. Apart from a deterministic model describing the growth rate of both the donor strain and the resistant recipient strain a stochastic model was developed that is particularly useful when low numbers of mutants are formed.

Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h−1) and low dilution rate (0.012 h−1) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.

Microorganism-based bioplasticizer for cementitious materials

A plasticizer resulting from mixed culture of microorganism with beneficial effect that co-exist in a liquid environment has been evaluated as potential admixture for cement. The bioplasticizer MEF (Microorganism-based bioplasticizer) contains fermentation products from liquid cultures of lactic bacteria, phototrophic bacteria and yeast of more than 80 different species. The material was tested in pastes and mortar using a rheology protocol, and compared with series made with one commercial superplasticizer (SP). Cement pastes were prepared with different dosage of MEF (4%, 6% and 8%). The experimental program included the evaluation of the plasticity index from the minicone test; viscosity through the Marsh cone, and setting time with the Vicat needle. Further, mortars with the same dosage were prepared and their rheology, water absorption and mechanical properties were assessed. Results indicate that MEF has plasticizing properties; however the effect is lower than commercial SPs. The main reasons appear to be the high dilution rate. Concentration through roto-evaporation brings about an increase in plasticizing properties. Separation by polar fractions shows that apparently the plasticizing principle is related to the presence of lactic acid. MEF seems to influence hydration of the aluminates, and in high dosages it may have some retarding effect. It has proven to contribute to decrease viscosity of cement pastes and no major differences in performance were found with commercial SPs. Tests carried out in mortars confirm the results obtained in pastes. Mortars made with MEF show similar mechanical properties than those of the reference.

Effect of defocus manner on laser cladding of Fe-based alloy powder

This investigation was focused on the influences of defocus manner and composite powders on laser cladding. Combining with the transmission characteristic of parallel Gaussian laser beam through a focusing lens, the energy distribution of the cross section of the laser beam in positive and negative defocused zone was analyzed and also verified by laser dotting ablation. Comparing with positive defocus, the negative defocus is an effective way for high quality laser cladding providing more advantages. Therefore, the negative defocus method is selected to solve the problem of high dilution rate and pores in the overlapping coating. The study showed that by adjusting the process parameters, a coating with low dilution and free of pores can be achieved easily in the negative defocus. Finally, the microstructure and microhardness of Fe-based alloy mixed with 5wt.% Cr3C2 coating were analyzed.

Continuous lignocellulosic ethanol production using Coleus forskohlii root hydrolysate

Root biomass of Coleus forskohlii obtained after extraction of forskolin constitutes more than 90% of the raw material rich in carbohydrates that could be used as a substrate for the production of bioethanol. Ethanol production from this waste biomass was optimized in batch and continuous fermentation. The maximum ethanol concentration of 31.32g/l was obtained with batch fermentation. Continuous production of ethanol was carried out using wood chips immobilized cells of Saccharomyces cerevisiae in packed bed reactor. The maximum ethanol concentration of 34.25g/l was obtained with nitrogen supplement and aeration as compared to 33.57g/l without supplement and aeration at 0.1 (1/h) dilution rate showing no effect of aeration and nitrogen at low dilution rate. The maximum ethanol productivity (15.88g/lh) was obtained at a dilution rate of 1 (1/h) with nitrogen supplement and aeration whereas ethanol productivity (13.48g/lh) was obtained at a dilution rate of 0.75 (1/h) without nitrogen supplement and aeration showing promising effect of nitrogen and aeration at high dilution rate. Immobilized column reactor was useful for the production of bioethanol, and also suggests efficient utilization of C. forskohlii root biomass for the production of bioethanol.

Hydrocarbon production in high density Botryococcus braunii race B continuous culture

Continuous cultures of Botryococcus braunii race B were maintained at photosynthetic cell densities as high as 20 g dry weight per liter for up to 3 months. Growth associated triterpene hydrocarbon accumulation was nearly constant at 22.5% of dry weight for a range of growth rates maintained by daily replacement of 5-15% of the respective cultures. The ability to achieve high cell concentrations and oil levels of roughly 5 g triterpene oil/L resulted from a combination of high light (∼ 1/4 full sun for 15 h/day) and replenishing stoichiometrically balanced growth medium. Due to light-limited growth conditions, cell concentration dropped nearly linearly with increased dilution rate. This reduction in cell number resulted in increased productivity per cell at higher dilution rates and was accompanied by a dramatic increase in algae colony size from 0.09 to 0.343 mm at high dilution rate. This change in colony size resulted in an equally dramatic change in optical density (OD) per gram dry weight, which precluded use of simple correlations of OD and cell concentration. A trickle-film photobioreactor was also demonstrated as a scalable approach to achieving these ultra-high cell concentrations. Additional media analysis revealed a steady increase in photobioreactor conductivity suggesting an accumulation of ions may be the reason for rapid culture crash and washout observed at all dilution rates after several months of continuous operation. The volumetric productivity of 22.5 mg oil/L/photo-h reported here is more than an order of magnitude higher than previous reports for B. braunii race B, reflecting the high cell densities used in this work and substantiating a higher metabolic rate for B. braunii race B than previously surmised from its relatively long doubling times.

Mixotrophic growth of Phaeodactylum tricornutum on fructose and glycerol in fed-batch and semi-continuous modes

Mixotrophic cultures of Phaeodactylum tricornutum were carried out in bubble columns using fructose and glycerol in indoor fed-batch and semi-continuous modes. In the fed-batch cultures, different nutrient-addition strategies, combined with stepwise increments in the light intensity, were assayed. It was found that glycerol promoted significantly higher biomass productivity than fructose. A glycerol-induced photoinhibition that arrested the growth of P. tricornutun was also observed. As this was considered a limitation as regards transferring the fed-batch mode to outdoor conditions, this information was used to culture P. tricornutum in semi-continuous mode. Similar glycerol-induced photoinhibition was not observed in these cultures, even at highest dilution rates. Although the highest biomass (1.5 g L(-1) d(-1)) and EPA (40 mg L(-1) d(-1)) productivities found in the semi-continuous cultures were lower than those obtained photoautotrophically in outdoor photobioreactors, the findings showed that semi-continuous mode was an excellent candidate for transferring mixotrophic culture to an outdoor setting.

Improving hydrolysis of food waste in a leach bed reactor

This paper examines the rate of degradation of food waste in a leach bed reactor (LBR) under four different operating conditions. The effects of leachate recirculation at a low and high flow rate are examined with and without connection to an upflow anaerobic sludge blanket (UASB). Two dilution rates of the effective volume of the leach bed reactors were investigated: 1 and 6 dilutions per LBR per day. The increase in dilution rate from 1 to 6 improved the destruction of volatile solids without connection to the UASB. However connection to the UASB greatly improved the destruction of volatile solids (by almost 60%) at the low recirculation rate of 1 dilution per day. The increase in volatile solids destruction with connection to the UASB was attributed to an increase in leachate pH and buffering capacity provided by recirculated effluent from the UASB to the leach beds. The destruction of volatile solids for both the low and high dilution rates was similar with connection to the UASB, giving 82% and 88% volatile solids destruction respectively. This suggests that the most efficient leaching condition is 1 dilution per day with connection to the UASB.

Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture

In this study, a continuous culture system was applied to mammalian cells on large scale, and polyethyleneimine (PEI) mediated transient gene expression (TGE). PEI MAX 40,000 was chosen as a superior reagent from three types of PEI. The cell cycle distribution of cells in batch and continuous cultures was determined, in which the effects of cell cycle distribution on transfection efficiency, post-transfection proliferation and recombinant prothrombin expression were evaluated. Compared with cells from end-log and plateau phase in batch culture, cells from mid-log phase possessed a larger fraction of S and G2/M phase cells and a smaller fraction of G1 phase cells. In the continuous culture, the fraction of cells in the S and G2/M phases increased and the fraction of cells in the G1/G0 phase decreased with increasing dilution rates. Cells from the continuous culture run at highest dilution rate had excellent proliferation, transfection efficiency and protein expression. These results were confirmed by transfecting cells synchronized to different phases. The G2/M arrested cells exhibited a nearly 10-fold increase in recombinant human prothrombin production relative to that of non-dividing cells. The use of continuous culture for large scale transfection demonstrated a better cell physiological state for TGE process.

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r=0.8–0.9) and dilution (up to 0.48h−1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160g/l at r=0.9 and dilution rate of 0.2h−1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5g/lh−1 could be achieved with feeding concentrations of 80–200g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48g/lh−1.

Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability

The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies.

Impact of Furfural on Rapid Ethanol Production Using a Membrane Bioreactor

A membrane bioreactor was developed to counteract the inhibition effect of furfural in ethanol production. Furfural, a major inhibitor in lignocellulosic hydrolyzates, is a highly toxic substance which is formed from pentose sugars released during the acidic degradation of lignocellulosic materials. Continuous cultivations with complete cell retention were performed at a high dilution rate of 0.5 h−1. Furfural was added directly into the bioreactor by pulse injection or by addition into the feed medium to obtain furfural concentrations ranging from 0.1 to 21.8 g L−1. At all pulse injections of furfural, the yeast was able to convert the furfural very rapidly by in situ detoxification. When injecting 21.8 g L−1 furfural to the cultivation, the yeast converted it by a specific conversion rate of 0.35 g g−1 h−1. At high cell density, Saccharomyces cerevisiae could tolerate very high furfural levels without major changes in the ethanol production. During the continuous cultures when up to 17.0 g L−1 furfural was added to the inlet medium, the yeast successfully produced ethanol, whereas an increase of furfural to 18.6 and 20.6 g L−1 resulted in a rapidly decreasing ethanol production and accumulation of sugars in the permeate. This study show that continuous ethanol fermentations by total cell retention in a membrane bioreactor has a high furfural tolerance and can conduct rapid in situ detoxification of medium containing high furfural concentrations.

Effect of the dilution process on the attenuation of contaminants in aquifers

This paper discusses the effects of the dilution process on levels of aquifer contamination attenuation, developing analytical equations for application in theoretical and practical cases. The key finding is that, in aquifers, the dilution process causes a reduction of the contaminant concentration. Analytical equations for the assessment of the contaminant attenuation capacity, by means of the dilution process in an aquifer, are put forward. In particular, the attenuation of the contaminant concentration in an aquifer, and thus the dilution, is described by the volumetric flow rate per unit width perpendicular to the flow direction (q u). Moreover, a classification of q u was developed, considering six classes of attenuation capacity due to dilution. For the equations validation, nitrate contamination attenuation due to dilution was analysed in two sample areas in Piedmont (North-West Italy). The results confirmed the hypothesis, evidencing high nitrate concentrations in areas with low q u values, connected to low dilution rate of the contaminant in the groundwater; vice versa, low nitrate concentrations were evidenced in areas with high q u values, due to a high nitrate dilution rate.

Quantitative physiology of Lactococcus lactis at extreme low‐growth rates

This paper describes the metabolic adaptation of Lactococcus lactis during the transition from a growing to a non-growing state using retentostat cultivation. Under retentostat cultivation, the specific growth rate decreased from 0.025 h(-1) to 0.0001 h(-1) in 42 days, while doubling time increased to more than 260 days. Viability of the overall culture was maintained above 90% but included approximately 20% damaged cells, which had lost their colony forming capacity on solid media. Although culture biomass and viability had reached a steady-state after 14 days of retentostat cultivation, the morphology of the cells changed from coccus-to-rod shape at later stages of retentostat cultivation, by which the cell's surface to volume ratio was estimated to increase 2.4-fold. Furthermore, the metabolic patterns switched between homolactic and mixed-acid fermentation during the retentostat cultivation. Retentostat cultivation enabled the calculation of accurate substrate- and energy-related maintenance coefficients and biomass yields under non-growing conditions, which were in good agreement with those calculated by extrapolation from chemostat cultivations at high dilution rates. In this study, we illustrate how retentostat cultivation allows decoupling of growth and non-growth associated processes in L. lactis, enabling the analysis of quantitative physiological responses of this bacterium to near zero-specific growth rates.

Decolourization of Amaranth dye by bacterial biofilm in batch and continuous packed bed bioreactor

The aim of this study was to exploit the bacterial biofilms to remove dyes from industrial effluents. Biofilms of strains AK1, AK2, VKY1 and a consortium on sheep bone chips were examined in batch, repeated batch and continuous packed bed bioreactor. Biofilms are more efficient for decolourization of Amaranth dye at three different dye concentrations (200, 400, and 600 mg l−1). 100% decolourization of Amaranth dye was observed even at higher concentrations (400 and 600 mg l−1) by all the tested biofilms in 24 h than that of corresponding free cells. The biofilms were superior over those of free cells and could be reused for more than 18 repeated cycles. In a packed bed bioreactor, biofilms could be operated with much higher dilution rates and at lower hydraulic retention time. Further, the decolourization of dye was confirmed by UV–visible spectrophotometer, TLC and HPLC analysis of Amaranth dye degradation products from packed bed bioreactor effluent.

Steady-state analysis of the Anaerobic Digestion Model No. 1 (ADM1)

The steady-state behavior of the Anaerobic Digestion Model No. 1 (ADM1) with respect to dilution rate and substrate concentration is analyzed in this study. Thereby, up to ten coexisting steady-state solutions are observed under the same operating conditions. The parameter region of a methane producing operation is limited regarding high dilution rates as well as low or high substrate concentrations. The underlying mechanisms causing those limits are investigated in detail, and the common core is identified, namely a positive feedback loop between growth of acetate degraders and acetate itself. The difference lies in the activation mechanisms of this feedback loop, which differs in the three investigated cases.