A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity

A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.     

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.     

Stability of the analytical solution of Penna model of biological aging

There are some analytical solutions of the Penna model of biological aging; here, we discuss the approach by Coe et al. (Phys. Rev. Lett. 89, 288103, 2002), based on the concept of self-consistent solution of a master equation representing the Penna model. The equation describes transition of the population distribution at time t to next time step (t + 1). For the steady state, the population n(a, l, t) at age a and for given genome length l becomes time-independent. In this paper we discuss the stability of the analytical solution at various ranges of the model parameters—the birth rate b or mutation rate m. The map for the transition from n(a, l, t) to the next time step population distribution n(a + 1, l, t + 1) is constructed. Then the fix point (the steady state solution) brings recovery of Coe et al. results. From the analysis of the stability matrix, the Lyapunov coefficients, indicative of the stability of the solutions, are extracted. The results lead to phase diagram of the stable solutions in the space of model parameters (b, m, h), where h is the hunt rate. With increasing birth rate b, we observe critical b ₀ below which population is extinct, followed by non-zero stable single solution. Further increase in b leads to typical series of bifurcations with the cycle doubling until the chaos is reached at some b _c. Limiting cases such as those leading to the logistic model are also discussed.     

Relationships between soil potassium supply characteristics based on soil solution concentration and buffer power and field responses of winter wheat and maize

The relationship between soil K supply, characterised by the soil solution K concentration and the soil K buffer power, and plant K status was investigated for field grown crops. The study was carried out in 15 K fertilisation trials with maize and wheat covering a wide range of agricultural soils and K level. Soil K buffer power was obtained through sorption–desorption curves. For each trial the critical K concentration in the soil solution was deduced from the relationship between the K concentration in the soil solution and the K concentration in shoot tissue water, which was considered as a relevant indicator of the plant K status. At sufficient K levels, the maximal K concentration in the shoot tissue water of maize (145 mM) was lower than that of wheat (175 mM) but the percentages of the critical K concentration in plant tissue water to this maximal K concentration were similar for the two plant species with an average of 75%. The critical K concentration in the soil solution varied between soils. However, a close correlation was found between the critical K concentration in soil solution and the inverse of the soil buffer power at this concentration (r ²=0.981) or the inverse of the square of buffer power (r ²=0.989). On a wide range of soils and field conditions, these two indicators were more able to account for K availability and plant response than exchangeable K content or K saturation ratio of the CEC.     

Simulating the rheology of surfactant solution using dissipative particle dynamics

Reverse non-equilibrium (RNE) method is applied to investigate the rheological properties of surfactant solutions by using dissipative particle dynamics algorithm. Results show that the surfactant solutions exhibit a shear-thinning behaviour. With the strengthening of shear, the worm-like micelles are gradually oriented in the x direction and then broken up into small spherical micelles. The process is also shown by the decrease of viscosity, which starts quickly, levels off for moderate shear rates and speeds up again over a critical value. We find that this critical value is independent of surfactant concentration. Compared with the shear flow method, the RNE method is credible for moderate shear rate and has more computational efficiency.     

RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.     

The effects of rumen fluid on the in vitro aflatoxin binding capacity of different sequestering agents and in vivo release of the sequestered toxin

In an in vitro experiment, aluminosilicates (Atox^® and Novasil™ Plus) and a yeast cell wall derivate (Mycosorb^®) were used as sequestering agents (SAs) to verify their capacity for binding aflatoxin B1 (AFB1) in vitro. SAs were individually mixed at three different ratios with AFB1 (1:5000, 1:50,000 and 1:500,000, w/w) in water (CTR), rumen fluid from a lactating cow with a low rumen pH (LRS) or rumen fluid from a dry cow with a high rumen pH (HRS), and then used in a 3 × 3 × 3 factorial arrangement of a completely randomized design. At the 1:500,000 AF:SA ratio Atox^® and Novasil™ Plus sequestered over 0.87 and 0.98 of the AFB1 in the CTR and rumen solutions (LRS and HRS), respectively. This efficacy decreased when the amount of clays was reduced, with higher values (P<0.001) for Atox^® compared with Novasil™ Plus (0.50 vs. 0.28 in CTR; 0.58 vs. 0.16 in LRS and 0.44 vs. 0.27 in HRS). Mycosorb^® had a lower sequestering efficacy (P<0.001) in all of tested experimental conditions, with 0.34 being the maximum value obtained in the CTR solution.     

Function and solution structure of the Arabidopsis thaliana RALF8 peptide

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with ¹⁵N and ¹³C for NMR analysis and obtained near complete ¹H, ¹³C, and ¹⁵N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.     

嗜酸氧化亚铁硫杆菌生长动力学方程的应用

基于Monod模型推导出了A．f的生长动力学方程模型,采用Gauss-Newton算法确定了在不同初始条件下细菌生长的动力学参数,即最大比生长速率‰、Monod常数K及R0。通过在不同初始条件下细菌生长特性的研究,得到了相应初始生长条件下以限制性底物亚铁离子浓度为表征的生长动力学方程,理论上揭示了动力学参数变化对细菌生长的影响规律,其中生长动力学方程的数值模拟与实验数据相吻合。