Mol Sucrose Research Articles

Salt and heat stress enhances hydrogen production in cyanobacteria.

Cyanobacteria are among the most suitable organisms for the capture of excessive amounts of CO2 and can be grown in extreme environments. In our research we use the single-celled freshwater cyanobacteria Synechococcus elongatus PCC7942 PAMCOD strain and Synechocystis sp. PCC6714 for the production of carbohydrates and hydrogen. PAMCOD strain and Synechocystis sp. PCC6714 synthesize sucrose when exposed to salinity stress, as their main compatible osmolyte. We examined the cell proliferation rate and the sucrose accumulation in those two different strains of cyanobacteria under salt (0.4M NaCl) and heat stress (35 0C) conditions. The intracellular sucrose (mol sucrose content per Chl a) was found to increase by 50% and 108% in PAMCOD strain and Synechocystis sp. PCC6714 cells, respectively. As previously reported, PAMCOD strain has the ability to produce hydrogen through the process of dark anaerobic fermentation (Vayenos D, Romanos GE, Papageorgiou GC, Stamatakis K (2020) Photosynth Res 146, 235-245). In the present study, we demonstrate that Synechocystis sp. PCC6714 has also this ability. We further examined the optimal conditions during the dark fermentation of PAMCOD and Synechocystis sp. PCC6714 regarding H2 formation, increasing the PAMCOD H2 productivity from 2 nmol H2 h- 1 mol Chl a- 1 to 23 nmol H2 h- 1 mol Chl a- 1. Moreover, after the dark fermentation, the cells demonstrated proliferation in both double BG-11 and BG-11 medium enriched in NaNO3, thus showing the sustainability of the procedure.

Efficacy of Passion Fruit Cryopreservation Using Cryopotectant Agents

ABSTRACT This study aimed to evaluate the effects of cryoprotectant agents on Passiflora eichleriana, Passiflora crystallina and Passiflora nitida seeds undergoing cryopreservation. The experiment was conducted with non-cryoprotected or non-cryopreserved seeds (control), cryopreserved seeds without cryoprotectant and cryopreserved seeds treated with the following cryoprotectants: 1.73, 2.28, 2.60 or 2.71 mol glycerol; 0.37, 0.46, 0.54 or 0.61 mol sucrose; or 0.37, 0.72, 1.04 or 1.35 mol dimethylsulfoxide (DMSO). After treatment, the seeds were cryopreserved in liquid nitrogen (LN2) for 7 days and then defrosted in 37°C water for 5 min. The seeds were submitted to germination and vigor tests consisting of four replicates of 50 seeds. Percentage of germination (PE) and germination velocity index, percentage of emergence velocity index, shoot and root length, and shoot and root dry mass were evaluated. After cryopreservation, all of germination and vigor values were higher without the use of cryoprotectants. The cryoprotectant glycerol had a deleterious effect on seed viability and vigor for all Passiflora species. Glycerol inhibit seedling emergence and concentrations higher than 2.60 mol seed germination on P. cristalina. Higher concentrations of DMSO decrease seed viability and vigor in P. crystalina and P. nitida.

Catalytic production of 5-hydroxymethylfurfural from sucrose and molasses by aluminum chloride in green aqueous γ-valerolactone system

5-Hydroxymethylfurfural (HMF) is a promising platform chemical for production of value-added chemicals and fuels. Conversion of abundantly available and cheap sucrose for HMF synthesis was systematically investigated in the present study. Results showed that Lewis acid AlCl3 is effective in catalytic transformation of sucrose. Combining AlCl3 with different Bronsted acids, no matter whether they were inorganic acids (HCl, H2SO4, and H3PO4) or organic acids (malic acid, succinic acid, and oxalic acid), or variation of ratio of Lewis acid/Bronsted acid represented by AlCl3/HCl, did not change the obtained maximum HMF yields compared with AlCl3 alone, but shortened the time required to reach the maximum HMF yield. Sucrose conversion in a green reaction media of aqueous γ-valerolactone (GVL) solvent resulted into comparable HMF yields with dimethyl sulfoxide (DSMO)-water system. Addition of monovalent salt NaCl and divalent salt Na2SO4 produced opposite effect with Cl− promoting while SO42− inhibiting HMF production. Furthermore, HMF synthesis from constituent monosaccharides of sucrose and molasses, a by-product from sugar manufacturing process, was also carried out and it was found that an equivmole mixture of glucose and fructose gave rise to almost identical HMF yield to the same mole of sucrose. Molasses, even after chemical pretreatment for removal of certain amount of inorganic salts and color substances, was significantly inferior to synthetic molasses in terms of HMF formation, indicating the complexity and difficulty in chemical valorization of molasses for production of platform molecules.

A fiber optic sensor based on Mach‐Zehnder interferometer structure for food composition detection

AbstractIn this article, fiber optic sensor (FOS) has been employed for food composition detection by using a Mach–Zehnder Interferometer (MZI) structure. The Single Mode–Multimode–Single Mode configuration with varied length of the sensing region with 4 cm and 8 cm were designed and analyzed. The FOS has been developed by using a fusion arc splicing technique. The sensor device has been tested with the major components of food compositions such as water, 1.0 mol sucrose solution and oil with the refractive index (RI) of 1.333, 1.384, and 1.464, respectively. The sensor device experienced a wavelength redshift as the RI increases. Meanwhile, it is also observed that the sensitivity of FOS is linearly proportional to the length of the sensor region. The best sensitivity achieved is 4.413 nm/RIU for 8 cm length of sensor region. The findings of this article can serve as a reference for improved sensitivity by manipulating the sensor's length.

Comparison between Two Cryo-devices for Vitrification of Immature Oocytes of Indigenous Zebu Cows in Bangladesh

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen (LN2). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in 50 μl droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at 39°C with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop (47.1±6.9%) than that of French mini straw (15.9±12.5%). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) (84.5±14.2%) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes

Cryoprotectant‐free vitrification of human spermatozoa in new artificial seminal fluid

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5mg/mL human serum albumin and 0.25mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09±10.33 vs. 36.80±13.75), grade A motility (36.59±11.40 vs. 16.41±11.24), and normal morphology (18.74±8.35 vs. 11.85±5.84) and viability (68.22±10.83 vs. 60.86±11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p=0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p<0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p<0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.

In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose

Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to15 enzymes under mild reaction conditions (i.e. 37°C and atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmol of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.

A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos

The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures.A new vitrification method using P10 and PEPeS was tested using rat embryos. The survival rate of vitrified embryos after exposure to P10 for 120, 300, or 600s ranged from 95.9% to 98.3%. The fetal developmental rate ranged from 57.7% to 65.2%, which was not significantly different from that of fresh embryos. The experimental results indicated that vitrification using a combination of P10 and PEPeS was suitable for cryopreservation of rat early stage embryos.

Sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts

Sucrose included in freezing medium causes embryos to shrink by losing intracellular water, so that intracellular ice formation is reduced and embryos can be frozen rapidly. Blastocyst consists of trophoblast and inner cell mass. It may be considered that the artificial shrunken blastocyst is not embryo but tightly packed somatic cells. This study was carried out to examine the necessary of sucrose addition to vitrification solution for vitrifying the artificial shrunken mouse blastocysts. Laboratory study using mice blastocyst. To investigate the toxicity of vitrification solutions and the availability of artificial shrinkage technique with ICSI pipette, blastocoel fluid was aspirated in the expanded mouse blastocysts using ICSI pipette and shrunken blastocysts were equilibrated in EBS1 and EBS2 for 1.5 min at room temperature, reapectively. Then the equilibrated blastocysts were exposed to vitrification solution for maximum of 20 sec. Two vitrification solutions with and without sucrose were prepared. Control and treatment were composed of 25%glycerol + 25% ethyleneglycol and 25%glycerol + 25%ethyleneglycero +0.5 mol/l sucrose, respectively. Assisted hatching was performed by partial zona dissection method using assisted hatching pipette after thawing procedure or rehydration. Rates of re-expanding and hatching were examined after culture for 6h. Re-expanding rate of mouse blastocyst exposed to VS(without and with 0.5 mol sucrose) were not different in G25E25(98%) and G25E25S0.5(92%) (P>0.05) and hatching rate was higher in G25E25(95%) than in G25E25S0.5(88%) but did not differ in two treatments (P>0.05). Re-expanding rate of mouse blastocyst vitrified in G25E25 and G25E25S0.5 was 95% and 90%, respectively (P=0.05), and hatching rate was higher in G25E25(90%) than in G25E25S0.5(76%) (P>0.05). Our results showed that sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts.

The Effect of Biomass Immobilization Support Material and Bed Porosity on Hydrogen Production in an Upflow Anaerobic Packed-Bed Bioreactor

The aim of this study was to investigate the effect of the support material used for biomass attachment and bed porosity on the potential generation of hydrogen gas in an anaerobic bioreactor treating low-strength wastewater. For this purpose, an upflow anaerobic packed-bed (UAPB) reactor fed with sucrose-based synthetic wastewater was used. Three reactors with various support materials (expanded clay, vegetal coal, and low-density polyethylene) were operated for hydraulic retention time (HRT) of 0.5 and 2 h. Based on the results obtained, three further reactors were operated with low-density polyethylene as a material support using various bed porosities (91, 75, and 50 %) for an HRT of 0.5 h. The UAPB reactor was found to be a feasible technology for hydrogen production, reaching a maximum substrate-based hydrogen yield of 7 mol H₂ mol(-1) sucrose for an HRT of 0.5 h. The type of support material used did not affect hydrogen production or the microbial population inside the reactor. Increasing the bed porosity to 91 % provided a continuous and cyclic production of hydrogen, whereas the lower bed porosities resulted in a reduced time of hydrogen production due to biomass accumulation, which resulted in a decreasing working volume.

Effect of effluent recycle ratio in a continuous anaerobic biohydrogen production system

Abstract The effect of effluent recycle on hydrogen production in an anaerobic continuous stirred tank reactor (CSTR) was investigated. The CSTR was fed on sucrose (20 g chemical oxygen demand (COD)/L) and at hydraulic retention time (HRT) of 12 h. The pH and temperature were regulated around 6.7 and 35 °C, respectively. The effluent was then recycled at recycle ratios of 0, 0.2, 0.4, 0.6, 0.8 and 1.0. When the recycle ratios increased, the volatile suspended solid (VSS) concentration also increased with a peak of 14.2% at the recycle ratio 0.4 compared with the VSS 3.52 g/L without effluent recycle. However, a drastic drop in hydrogen production performance was observed with increased recycle ratio. The strategy of increasing organic loading rate by increasing the substrate concentration and shorting the HRT was then applied keeping a recycle ratio of 0.2. VSS concentration increased with increasing substrate concentration but as the HRT was shortened from 12 h to 2 h stepwise, VSS concentration increased first and then decreased at HRT 8 h. The maximal hydrogen production was obtained at sucrose concentration of 20 g COD/L and HRT 6 h (organic loading rate (OLR) 66.7 g COD/L-d) with recycle ratio 0.2. The hydrogen yield, hydrogen production rate and specific hydrogen production rate values also peaked at the same conditions (sucrose 20 g COD/L and HRT 6 h) with 3.88 mol H 2 /mol sucrose, 807 mmol H 2 /L-d and 244.3 mmol H 2 /g VSS-d, respectively. This study showed peak hydrogen production performance at OLR 66.7 g COD/L-d in the recycled continuous anaerobic biohydrogen production system by controlling HRT. Thus, effluent recycling at the optimum ratio along with optimum HRT and substrate concentration can maximize the hydrogen production performance in an anaerobic biohydrogen production system.

The influence of the degree of back-mixing on hydrogen production in an anaerobic fixed-bed reactor

This paper reports on results obtained from experiments carried out in an acidogenic anaerobic reactor aiming at the optimization of hydrogen production by altering the degree of back-mixing. It was hypothesized that there is an optimum operating point that maximizes the hydrogen yield. Experiments were performed in a packed-bed bioreactor by covering a broad range of recycle ratios (R) and the optimum point was obtained for an R value of 0.6. In this operating condition the reactor behaved as 8 continuous stirred-tank reactors in series and the maximum yield was 4.22 mol H2 mol sucrose−1. Such optimum point was estimated by deriving a polynomial function fitted to experimental data and it was obtained as the conjugation of three factors related to the various degrees of back-mixing applied to the reactor: mass transfer from the bulk liquid to the biocatalyst, liquid-to-gas mass transfer and the kinetic behavior of irreversible reactions in series.

Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone

Ceramic ring and pumice stone were used as a support matrix for the enhancement of biohydrogen production in immobilized cell culture systems. The reactors were continuously operated for the hydrogen fermentation using sucrose as the major carbon source at varying hydraulic retention times (HRT) as an important operational factor. In terms of volumetric hydrogen production, the best value was obtained with ceramic ring at 1.5 h HRT (2.98 l H 2/l/d), on the other hand, the pumice stone packed reactor resulted in 30% less volumetric hydrogen production (2.28 l H 2/l/d) at two fold longer retention time (HRT 3 h). It was demonstrated that volumetric hydrogen production with the immobilized bioreactor configurations was 6 fold better than the suspended culture bioreactor configuration (CSTR). Furthermore, up to 4 mol and 5 mol hydrogen yields per mole of sucrose used (which are 62.5% and 50% of the theoretical values) were achieved by pumice stone and ceramic ring packed reactors, respectively, whereas suspended culture system yielded only 0.5 mol H 2/mol sucrose.

Succinate production from sucrose by metabolic engineered escherichia coli strains under aerobic conditions

Two metabolically engineered E. coli strains HL2765k and HL27659k, while capable of producing succinate from glucose with high yields, are not able to grow and produce succinate on sucrose. Consequently, the pUR400 plasmid containing scrK, Y, A, B, and R genes was introduced into HL2765k and HL27659k, respectively. Shake flask culture studies showed that the resulting strains can utilize sucrose; the strain HL2765k pUR400 and HL27659k pUR400 can produce succinate aerobically with a molar yield of 0.78 ± 0.02 mol/mol and 1.35 ± 0.13 mol/mol, respectively. On introduction of the plasmid pHL413, which encodes the heterologous pyruvate carboxylase (PYC) from Lactococcus lactis, the molar succinate yield increased to 1.60 ± 0.01 mol of succinate per mole of sucrose by the HL2765k pUR400 pHL413 strain and to 1.84 ± 0.10 by the HL27659k pUR400 pHL413 strain. In aerobic batch bioreactor studies, the succinate production rate was faster, and succinate production reached 101.83 mM with a yield of 1.90 when dissolved oxygen (DO) was controlled at 40 ± 7%. In addition, the results showed that DO had an important effect on succinate production by influencing PYC activity. This work demonstrates the possibility of producing succinate aerobically using sucrose as the carbon source.

Sequential dark–photo fermentation and autotrophic microalgal growth for high-yield and CO 2-free biohydrogen production

Dark fermentation, photo fermentation, and autotrophic microalgae cultivation were integrated to establish a high-yield and CO 2-free biohydrogen production system by using different feedstock. Among the four carbon sources examined, sucrose was the most effective for the sequential dark (with Clostridium butyricum CGS5) and photo (with Rhodopseudomonas palutris WP3-5) fermentation process. The sequential dark–photo fermentation was stably operated for nearly 80 days, giving a maximum H 2 yield of 11.61 mol H 2/mol sucrose and a H 2 production rate of 673.93 ml/h/l. The biogas produced from the sequential dark–photo fermentation (containing ca. 40.0% CO 2) was directly fed into a microalga culture ( Chlorella vulgaris C–C) cultivated at 30 °C under 60 μmol/m 2/s illumination. The CO 2 produced from the fermentation processes was completely consumed during the autotrophic growth of C. vulgaris C–C, resulting in a microalgal biomass concentration of 1999 mg/l composed mainly of 48.0% protein, 23.0% carbohydrate and 12.3% lipid.

Enhancing fermentative hydrogen production from sucrose

This study evaluated the hypothesis that fermentative hydrogen production from organic-rich feedstock could be enhanced by supplementing with waste materials such as cattle manure that could provide nutritional needs, buffering capacity, and native hydrogen-producing organisms. This hypothesis was tested in batch reactors fed with sucrose blended with cattle manure run at 25 degrees C without any nutrient supplements, pH adjustments, buffering, or gas-sparging. Hydrogen production rates in these reactors ranged 16-30 mL H(2)/g DeltaCOD-day, while hydrogen content in the biogases ranged 50-59%. Compared to literature studies conducted at higher temperatures, hydrogen yields found in this study at 25 degrees C were higher in the range of 3.8-4.7 mol H(2)/mol sucrose added, with higher positive net energy yields (>14 kJ/L). This study demonstrated that cattle manure as a supplement could not only provide hydrogen-producing seed, nutritional needs, and buffering capacity, but also increase hydrogen yield by approximately 10%, improving the economic viability of fermentative biohydrogen production from sugary wastes.

Simultaneous production of 2,3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge

A Klebsiella sp. HE1 strain isolated from hydrogen-producing sewage sludge was examined for its ability to produce H 2 and other valuable soluble metabolites (e.g., ethanol and 2,3-butanediol) from sucrose-based medium. The effect of pH and carbon substrate concentration on the production of soluble and gaseous products was investigated. The major soluble metabolite produced from Klebsiella sp. HE1 was 2,3-butanediol, accounting for over 42–58% of soluble microbial products (SMP) and its production efficiency enhanced after increasing the initial culture pH to 7.3 (without pH control). The HE1 strain also produced ethanol (contributing to 29–42% of total SMP) and a small amount of lactic acid and acetic acid. The gaseous products consisted of H 2 (25–36%) and CO 2 (64–75%). The optimal cumulative hydrogen production (2.7 l) and hydrogen yield (0.92 mol H 2 mol sucrose −1) were obtained at an initial sucrose concentration of 30 g COD l −1 (i.e., 26.7 g l −1), which also led to the highest production rate for H 2 (3.26 mmol h −1 l −1), ethanol (6.75 mmol h −1 l −1) and 2,3-butanediol (7.14 mmol h −1 l −1). The highest yield for H 2, ethanol and 2,3-butanediol was 0.92, 0.81 and 0.59 mol mol-sucrose −1, respectively. As for the overall energy production performance, the highest energy generation rate was 27.7 kJ h −1 l −1 and the best energy yield was 2.45 kJ mol sucrose −1, which was obtained at a sucrose concentration of 30 and 20 g COD l −1, respectively.

Hydrogen Production by Anaerobic Microbial Communities Exposed to Repeated Heat Treatments

Biological hydrogen production by anaerobic mixed communities was studied in laboratory-scale bioreactors using sucrose as the substrate. A bioreactor in which a fraction of the return sludge was exposed to repeated heat treatments performed better than a control bioreactor without repeated heat treatment of return sludge and produced a yield of 2.15 moles of hydrogen per mole of sucrose, with 50% hydrogen in the biogas. Terminal restriction fragment length polymorphism analysis showed that two different Clostridium groups (comprised of one or more species) were dominant during hydrogen production. The relative abundance of two other non-Clostridium groups increased during periods of decreased hydrogen production. The first group consisted of Bifidobacterium thermophilum, and the second group included one or more of Bacillus, Melissococcus, Spirochaeta, and Spiroplasma spp.

Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing

Methods for accurate and efficient quantification of metabolic fluxes are desirable in plant metabolic engineering and systems biology. Toward this objective, we introduce the application of “bondomers”, a computationally efficient and intuitively appealing alternative to the commonly used isotopomer concept, to flux evaluation in plants, by using Catharanthus roseus hairy roots as a model system. We cultured the hairy roots on (5% w/w U– 13C, 95% w/w naturally abundant) sucrose, and acquired two-dimensional [ 13C, 1H] and [ 1H, 1H] NMR spectra of hydrolyzed aqueous extract from the hairy roots. Analysis of these spectra yielded a data set of 116 bondomers of β-glucans and proteinogenic amino acids from the hairy roots. Fluxes were evaluated from the bondomer data by using comprehensive bondomer balancing. We identified most fluxes in a three-compartmental model of central carbon metabolism with good precision. We observed parallel pentose phosphate pathways in the cytosol and the plastid with significantly different fluxes. The anaplerotic fluxes between phosphoenolpyruvate and oxaloacetate in the cytosol and between malate and pyruvate in the mitochondrion were relatively high (60.1 ± 2.5 mol per 100 mol sucrose uptake, or 22.5 ± 0.5 mol per 100 mol mitochondrial pyruvate dehydrogenase flux). The development of a comprehensive flux analysis tool for this plant hairy root system is expected to be valuable in assessing the metabolic impact of genetic or environmental changes, and this methodology can be extended to other plant systems.

The improvement in fertilizing ability of cryopreserved mouse spermatozoa using laser-microdissected oocytes.

Aim: The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa. Methods: C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed. Results: When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments. Conclusion: The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. (Reprod Med Biol 2006; 5: 249-253).