The potential of glycerol in freezing preservation of turbine oil-degrading bacterial consortium and the ability of the revised consortium to degrade petroleum wastes

The turbine oil (TuO)-degrading bacterial consortium Tank-2 (original Tank-2) was preserved as a glycerol stock at −80 °C from 2009 to 2012. Storage methods have been unavailable so far for any TuO-degrading bacterial consortia or isolates. To evaluate the usefulness of glycerol stock, the original Tank-2 consortium frozen in glycerol at −80 °C was thawed and then revived by repeated culture in mineral salts medium (MSM) containing 0.5% (w/w) TuO (revived Tank-2). The revived Tank-2 consortium exhibited a high activity to degrade TuO, which was equivalent to that of original Tank-2. It also degraded car engine oil, used car engine oil, Arabian light and Vityaz crude oils and TuO in wastewater. These results indicated that a glycerol stock at −80 °C was useful for storing Tank-2. PCR-denaturing gradient gel electrophoresis (DGGE) that targeted the V3 regions of 16S rRNA gene sequences showed that the DGGE band profiles of principal bacteria were significantly different between the original and revived Tank-2 consortia and between the revived Tank-2 culture grown in MSM containing TuO and that grown in MSM containing other types of petroleum products. This suggested that bacterial strains inherently residing in Tank-2 could adjust their compositions based on the storage and culture conditions.     

Optimization of the cryopreservation of biological resources,Toxoplasma gondii tachyzoites,using flow cytometry

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10⁶ tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me₂SO without incubation. A cooling rate of ∼1 °C/min to −80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.     

The death pathways in mussel larval cells after a freeze-thaw cycle

We analyzed cell viability, caspase activity, plasma membrane alterations and cell ultrastructure morphology to estimate the morphological and biochemical alterations that occur in bivalve molluscan cell cultures during cryopreservation. The use of 5% dymethyl sulfoxide as a cryoprotectant resulted in greater cell survival and a scarcity of destroyed cells lacking cytosol among dead cells. In this case, almost all cells died through necrosis or apoptosis, which appeared to increase in mussel cell cultures after a freeze-thaw cycle. Apoptosis was not a main death pathway in mussel cells, but it was induced in a significant part of these cells (up to 24%) immediately after thawing and depended mostly on the cryoprotectant used. Regardless of the type of the used cryoprotectant, we observed some nuclear aberrations in cells after freezing-thawing, such as few multipolar mitoses or the absence of a division spindle in mitotic cells. After analyzing different methods for assessing cell damage, the best results were obtained from optimal approaches that could provide information regarding the cell disruption level after freezing-thawing and could be considered for future studies.     

Cat preantral follicle survival after prolonged cooled storage followed by vitrification

The aim of this study was to investigate the impact of prolonged storage at 4 °C on survival of cat preantral follicles (PAFs) pre- and post-vitrification. Ovaries were obtained from 12 queens and transported at 4 ºC within 2–6 h. Parts of the ovaries were stored for an additional 24 h or 72 h. The ovarian cortex was dissected, analyzed for viability (neutral red - NR) and morphology (histology - HE and ultrastructural analysis by TEM) and vitrified. We used 2 mm biopsy punches to obtain equal size pieces as the experimental units. After NR assessment, each sample was fixed and embedded in paraffin for HE staining to determine the number of morphologically intact follicles. Another 2 mm piece of ovary was subjected to TEM. NR viability assessment and HE results showed a similar tendency with PAF survival postvitrification even after prolonged cooling at 24 h and 72 h. With TEM, integrity of mitochondria, plasma and basal membranes as well as the presence of pre-granulose cells of PAFs were documented postvitrification for the control group and 24 h prolonged storage group, but not after 72 h storage. Our results showed that cat PAFs can survive prolonged storage followed by vitrification. The described set of techniques are applicable towards creating a gamete bank for endangered feline species.     

Shifts in thermoregulatory strategy during ontogeny in harp seals (Pagophilus groenlandicus)

Heat balance can be difficult for young and/or small animals in polar regions because environmental conditions in combination with small body size or physiological immaturity can increase heat loss. We investigated how thermoregulatory patterns change with ontogeny in 5 age classes of harp seal (Pagophilus groenlandicus) from birth to post-molt to further understand the timing of thermoregulatory development in relation to their potential vulnerability to ongoing fluctuations in the extent and stability of Arctic pack ice. We measured changes in the amount, conductivity, and resistance of the seal pups׳ insulative layers (blubber and fur), the potential for endogenous heat-generation by shivering (muscle enzyme activity), and nonshivering thermogenesis (NST; brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression and mitochondrial density). There was no significant difference in blubber conductivity among age classes, though the amount of blubber insulation significantly increased from birth to weaning. Pelage conductivity was low (0.12±0.01 W m⁻¹ °C⁻¹) except in 9-day old pups (0.40±0.08 W m⁻¹ °C⁻¹); the significantly higher conductivity may signal the beginning of the molt, and this age group may be the most vulnerable to early water entry. Citrate synthase activity significantly increased (49.68±3.26 to 75.08±3.52 μmol min⁻¹ g wet weight⁻¹) in the muscle; however it is unlikely that increasing a single enzyme greatly impacts heat generation. BAT of younger pups contained UCP1, though expression and mitochondrial density quickly declined, and the ability of pups to produce heat via NST was lost by weaning. While total thermal resistance did not differ, neonatal and early nursing animals gained the majority of their thermal resistance from lanugo (82.5±0.03%); however, lanugo is not insulative when wet, and NST may be important to maintain euthermia and dry the coat if early immersion in water occurs. By late nursing, blubber seems sufficient as insulation (75.87±0.01% of resistance after 4 weeks), but high conductivity of fur may be responsible for retention of UCP1 expression. Weaned animals rely on blubber insulation, and no longer need NST, as wetted fur is no longer a threat to euthermia.     

Human sperm cryopreservation in cancer patients: Links with deprivation and mortality

Evidence is mounting for a relationship between human semen quality and environmental/lifestyle/socioeconomic factors including long term health outcomes such as mortality. The relationship between pre-freeze and post-thaw semen quality in cancer patients and these factors are unknown. Frozen semen from 217 cancer patients was thawed and analysed using a validated CASA method. Post-thaw quality was matched and compared with WHO semen analysis performed prior to storage. The English Indices of Deprivation 2010 were matched with patients and then examined for relationships with pre-freeze and post-thaw semen quality. There is a relationship between semen quality and deprivation in cancer patients. Compared with pre-freeze semen quality, post-thaw semen quality has a stronger relationship with deprivation. Sperm cryopreservation may have potential as a systemic health diagnostic test and is predictive of cancer patient mortality.     

Genetic factors in individual radiation sensitivity

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60 min of repair.Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p = 0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p = 0.039): B_CC = −1.06 (95%-CI: −1.16 to −0.96), B_CT = −1.02 (95%-CI: −1.11 to −0.93) and B_TT = −0.85 (95%-CI: −1.01 to −0.68). In both cases and controls, we observed significantly higher DNA basal damage (p = 0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p = 0.781). An alteration to DRC after 30 min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p = 0.055), but was the most significant finding in the sample of families (p = 0.009).Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.     

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.     

The oxidant-antioxidant equilibrium,activities of selected lysosomal enzymes and activity of acute phase protein in peripheral blood of 18-year-old football players after aerobic cycle ergometer test combined with ice-water immersion or recovery at room temperature

The goal of the study was to evaluate the effect of an aerobic exercise bout followed by ice-water immersion or recovery at room temperature on the redox state, activities of selected lysosomal enzymes and activity of α1-antitrypsin (AAT) in the blood of healthy sportsmen. Eleven amateur football players aged 18 were randomly assigned to two similar 30-min aerobic cycle ergometer tests followed by a recovery at room temperature (20 °C; Experiment 1) or ice-water immersion (3 °C, 5 min; Experiment 2). Peripheral blood was collected three times during both study experiments: before (baseline), as well as 20 and 40 min after the recovery or immersion. The concentrations of thiobarbituric acid reactive substances in blood plasma (plTBARS) and erythrocytes (erTBARS) were measured. The erythrocytic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were also determined. In the blood serum, the activities of acid phosphatase (AcP), arylsulphatase (ASA), cathepsin D (CTS D) and AAT were evaluated. The activities of AcP, ASA, CTS D and AAT changed similarly during both experiments. The GPx activity decreased 40 min after the exercise/recovery compared to the baseline activity and was lower than 40 min after the exercise/immersion. The exercise followed by the recovery or immersion had no significant effect on the serum lysosomal and AAT activities in the studied men. The exercise/recovery reduced the hydrogen peroxide concentration in the men's erythrocytes, however the exercise/immersion demonstrated the opposite effect.     

Pirfenidone inhibits cryoablation induced local macrophage infiltration along with its associated TGFb1 expression and serum cytokine level in a mouse model

PurposeTo investigate the effects of pirfenidone (PFD) on post-cryoablation inflammation in a mouse model.Materials and methodsIn this IACUC-approved study, eighty Balb/c mice were randomly divided into four groups (20/group): sham + vehicle, sham + PFD, cryoablation + vehicle, and cryoablation + PFD. For cryoablation groups, a 20% freeze rate cryoablation (20 s to less than −100 °C) was used to ablate normal muscle in the right flank. For sham groups, the cryoprobe was advanced into the flank and maintained for 20 s without ablation. PFD or vehicle solution was intraperitoneally injected (5 mg/kg) at days 0, 1, 2, 3, and then every other day until day 13 after cryoablation. Mice were euthanized at days 1, 3, 7, and 14. Blood samples were used for serum IL-6, IL-10, and TGFβ1 analysis using electrochemiluminescence and ELISA assays, respectively. Immunohistochemistry-stained ablated tissues were used to analyze macrophage infiltration and local TGFβ1 expression in the border region surrounding the cryoablation-induced coagulation zone.ResultsCryoablation induced macrophage infiltration and increased TGFβ1 expression in the border of the necrotic zone, and high levels of serum IL-6, peaking at days 7 (70.5 ± 8.46/HPF), 14 (228 ± 18.36/HPF), and 7 (298.67 ± 92.63), respectively. Animals receiving PFD showed reduced macrophage infiltration (35.5 ± 16.93/HPF at day 7, p < 0.01) and cytokine levels (60.2 ± 7.6/HPF at day 14, p < 0.01). PFD also significantly reduced serum IL-6 levels (p < 0.001 vs. all non-PFD groups).ConclusionsPFD mitigates cryoablation induced muscle tissue macrophage infiltration, increased IL-6 levels, and local TGFβ1 expression in a small animal model.     

Colorectal cancer risk and dyslipidemia: A case–cohort study nested in an Italian multicentre cohort

BackgroundDyslipidemia is an established risk factor for many diseases, but its effect on colorectal cancer risk is less clear. We investigated the association of colorectal cancer risk with plasma triglycerides, total, HDL, and LDL cholesterol in four Italian EPIC centers.MethodsWe conducted a case–cohort study on participants recruited to four Italian EPIC centers (Turin, Varese, Naples, and Ragusa; 34,148 subjects). A random subcohort of 850 subjects was obtained and 286 colorectal cancer cases were diagnosed. Triglycerides, total and HDL cholesterol were determined in plasma samples obtained at baseline and stored at −196 °C; LDL cholesterol was calculated. Hazard ratios (HR) with 95% confidence intervals (CI), adjusted for potential confounders, were estimated by Cox regression models using the Prentice method.ResultsThe highest tertiles of total (HR 1.66, 95%CI 1.12–2.45) and LDL cholesterol (HR 1.87, 95%CI 1.27–2.76) were associated with increased colorectal cancer risk compared to lowest tertiles. Risks were greater for men than women, and for postmenopausal than premenopausal women. Highest tertiles of total and LDL cholesterol were also significantly associated with increased risks of colon cancer, distal colon cancer, and rectal cancer, but not proximal colon cancer.ConclusionsOur findings suggest that high levels of total and LDL cholesterol increase colorectal cancer risk, particularly in men and postmenopausal women. However additional studies are needed to clarify the role of plasma lipids in these cancers, particularly in view of the conflicting findings of previous studies.     

Maintaining viability and characteristics of cholangiocarcinoma tissue by vitrification-based cryopreservation

Tumor tissue has great clinical and scientific value which relies highly on the proper preservation of primary materials. Conventional tumor tissue cryopreservation using slow-freezing method has yielded limited success, leading to significant cell loss and morphological damage. Here we report a standardized vitrification-based cryopreservation method, by which we have successfully vitrified and warmed 35 intrahepatic cholangiocarcinoma (ICC) tissues with up to 80% viability of the fresh tumor tissues. Cryopreserved ICC tissue could generate patient-derived xenografts (PDXs) with take rates of 68.2% compared to 72.7% using fresh tumor tissues. Histological and genetic analyses showed that no significant alterations in morphology and gene expression were introduced by this cryopreservation method. Our procedure may facilitate collection, long-time storage and propagation of cholangiocarcinoma or other tumor specimens for (pre)clinical studies of novel therapies or for basic research.     

Effect of cryopreservation on viability and growth efficiency of stromal-epithelial cells derived from neonatal human thymus

The thymus is the major site of T lymphocyte generation and so is critical for a functional adaptive immune system. Since, thymectomy is a component of neonatal surgery for congenital heart diseases, it provides great potential for collection and storage of thymic tissue for autologous transplantation. However, specific investigation into the optimum parameters for thymic tissue cryopreservation have not been conducted. In this research, we evaluated the effect of different cryoprotective media compositions, which included penetrating (Me₂SO, glycerol) and non-penetrating (dextran-40, sucrose, hydroxyethyl starch) components, on the viability and functionality of frozen-thawed human thymic samples to select an optimal cryoprotective medium suitable for long-term storage of thymic tissue and a stromal-epithelial enriched population. Our primary focus was on receiving, low-temperature storage, culturing and evaluation of thymic tissue samples from newborns and infants with congenital heart diseases, who had undergone thymectomy as a part of standard surgical procedure. Thus, this work builds the platform for autologous clinical intervention into the thymus-deficient patients with congenital heart diseases. From our data, we conclude that although there were no significant differences in efficiency of tested cryoprotective media compositions, the combination of Me₂SO and dextran-40 compounds was the most suitable for long-term storage both thymic cell suspensions and thymic fragments based on the viability of CD326⁺ epithelial cells and stromal-epithelial cell monolayer formation.     

Effect of resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality

Although vitrification is the current routine method for human embryo cryopreservation, it may cause detrimental effects. The aim of this study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or vitrification solutions (VS) with Resveratrol on the presence of apoptotic markers, reactive oxygen species (ROS) level, glutathione (GSH) content and relative gene abundance. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Zygotes were cultured in IVC medium supplemented with or without 0.5 μM Resveratrol (C^R, C⁻ respectively). On day 7, blastocysts were vitrified using the minimum volume vitrification method supplementing VS with (C⁻V^R, C^RV^R) or without (C^–V^-, C^RV⁻) 0.5 μM Resveratrol. After warming, embryonic quality parameters were evaluated. Survival rates were significantly lower in C^RV^R group compared with C^RV⁻ group, but no differences in hatching rate were observed between groups. Vitrification/warming process did not alter total cell number or the presence of apoptotic or dead cells, but C^RV⁻ and C^RV^R groups presented a significant increase in dead cells (P < 0.05 by ANOVA). Resveratrol supplementation in VS (C⁻V^R) restored GSH content (P < 0.05) to the level found in the C^R group. Vitrification/warming process significantly increased the expression of FOXO3A, PNPLA2, BCL2L1 and BAX genes (P < 0.05). Resveratrol addition to IVC medium or VS partially compensated this increase for FOXO3A and PNPLA2 (P < 0.05) but not for BCL2L1 and BAX. In conclusion, supplementation of IVC media or VS with 0.5 μM resveratrol may help embryos to partially restore the initial quality they had before the cryopreservation process.     

Effect of molybdenum on secondary metabolic process of glycyrrhizic acid in Glycyrrhiza uralensis Fisch.

A pot experiment was conducted to investigate into effects of molybdenum (Mo) on the secondary metabolic process of glycyrrhizic acid (GA). One-year-old seedlings were grown in pots with washed vermiculite and sand. Hoagland nutrition solution was irrigated with four concentrations: 0, 0.52, 5.2 and 10.4 mg L⁻¹. The accumulations of GA and its biosynthetic precursors (β-amyrin and squalene) and then expression of the key synthase (β-amyrin synthase, β-AS) were studied on 35, 70 and 105 d. In the early stage, that was on the 35 and 70 d, the contents of squalene and GA, and the expression of β-AS gene under 0.52 and 5.2 mg L⁻¹ Mo treatments were significantly higher than that under 0 and 10.4 mg L⁻¹ Mo. There was a contrary result of β-amyrin. However, the content of squalene under 0 mg L⁻¹ Mo was the highest on 105 d. Thus, it suggested an appropriate concentration of Mo could promote the accumulation of GA, by affecting the biosynthetic process of GA at a certain time. Practically, the time and amount of application of Mo on Glycyrrhiza uralensis should be the noted.     

Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli

To promote the decomposition of sugarcane bagasse (SCB) for conversion into value-added products and to reduce waste, the capability of fungal mixes (FMs) to degrade SCB was examined. A total of 169 isolates from SCB and non-SCB were categorized as thermotolerant and thermoresistant. Thirty-six fungal candidates were screened for the presence of polyphenol oxidase, endoglucanase (EDN) and xylanase (XLN) activities, and EDN and XLN activities were quantitated. Five identified isolates (Aspergillus flavus AG10; Aspergillus niger AG68 & NB23; and Penicillium citrinum AG93 & AG140) were selected as the best enzyme producers, and 15 moderately to highly xylolytic, cellulolytic and ligninolytic isolates were added to construct FMs. Using a Taguchi design, the top ten reducing sugar-producing FMs (no. 12 showed the maximum amount of reducing sugar, at 2.11 mg g⁻¹, followed by no. 7, 15, 2, 16, 11, 13, 6, 4, & 8) were selected as potential agents for decomposition durations of 1, 2 and 3 months. The maximum decrease in SCB materials compared with the control was generated by FM 6 (9.08% cellulose reduction); FM 13 (21.03% hemicellulose reduction); and FM 16 (9.21% lignin reduction). These results indicate the potential use of SCB as a substrate for synergistic FMs. These FMs could be applied to the large-scale composting of SCB and other related agricultural residues, thus improving the biological pretreatment of lignocellulose.