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Flax fiber is an important textile material with excellent antibacterial activity and moisture wicking. Degumming of flax roving is essential in determining flax-fiber quality. Traditional degumming requires a large amount of chemicals to process flax roving. This study aimed to reduce the chemical usage and achieve cleaner production during this process by applying microbial treatment to degum flax roving. Microbial degumming instead of acid pickling and NaClO2-bleaching steps can no longer use H2SO4 and NaClO2, and the amount of alkali can be reduced by 50%. By analyzing the compositional changes of the main steps in traditional degumming processes and microbial-treatment sample, the application of Bacillus subtilis HR5 can achieve a good degumming effect. Results of environmental scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffractometry analyses showed that the gum was remarkably reduced, which can be confirmed by the gum components. Overall, the breaking tenacity and antibacterial activity of fibers degummed by microbial treatment were better than those treated by traditional degumming. These findings demonstrated the feasibility of microbial treatment as a solution for flax roving degumming.

Ethnopharmacological relevance In traditional Chinese medicine, the aerial parts of Aeschynomene indica L. (AIL) have been used for wound healing, and to treat urinary tract infection, hepatitis, enteritis, dysentery, nyctalopia, conjunctivitis, urticaria, and furuncle. However, no scientific investigation has been conducted on its wound healing potential. Aim of the study To investigate the effects of AIL extract on wound healing, isolate the active constituent and reveal the possible mechanism of enhancing wound healing. Materials and methods The circular excision wound healing model was used to evaluate in vivo wound-healing activity. Hematoxylin and eosin staining was applied to assess inflammatory cells infiltration, angiogenesis, fibroblast proliferation, collagen synthesis, collagen remodeling, and skin appendages generation. Sirius red-picric acid staining was employed for quantitative analysis of the ratio of collagen I/III. Immunohistochemical staining for CD68, CCR7 (CD197), CD163, TGF-β1 and α-SMA was performed to determine macrophages phenotypes transition (M1-to-M2) and prove the scar-improving effect of AIL on wound healing. Results We successfully isolated the active constituent (Sub-Fr0.2) for wound healing from AIL extract, circular excision wound healing experiment and hematoxylin & eosin staining showed Sub-Fr0.2 has a significant promoting effect on wound healing. Results of sirius red-picric acid staining demonstrated a reduced ratio of collagen I/III in the Sub-Fr0.2 group as compared with the vehicle group. Immunohistochemical staining for CD68, CCR7 (CD197), and CD163 in the Sub-Fr0.2 group exhibited an elevated speed of macrophages transiting from M1 phenotype to M2 phenotype, when compared with the vehicle group. Besides, the expression of TGF-β1 and α-SMA were inhibited on wounds treated with the ointment containing Sub-Fr0.2. Conclusion Leaves of AIL and its active constituent (Sub-Fr0.2) effectively promoted wound healing and reduced scar formation, this efficacy might be exerted by accelerating macrophages phenotypes transition and inhibiting TGF-β1 and α-SMA expression.

Releasing saponins to aqueous phase from Dioscorea zingiberensis C. H. Wright tubers is an effective way to decrease water pollution in diosgenin industry. Aqueous saponins (AS) are unstable during storage and could be hydrolyzed under certain conditions. To preserve the maximum AS content, different storage methods were evaluated, and it was found that storage at −20 °C and storage after 80 °C drying could well maintain AS content stability. AS model solution system was then constructed and utilized to simulate storage process. Adding sodium dodecyl sulphate and boiling can both effectively ensure the stability of AS content, while acid shock deferred the decrease of AS content. AS content decreased after sequential effects of endogenous enzymes and microbial community from tubers in different phase during storage. Myroides, Enterococcus, Alcaligenes and Stenotrophomonas were enriched as storage proceeded. It was also concluded that multiple glycosidase and surfactants secreted by selectively enriched microbial community decreased AS content stability at late stage of storage. This study also provides a guideline for preventing the loss of glycoside bioactive constituents in other industrial crops during storage.

This study provides the presence of carbonic anhydrase (CA) activity in waters of the Yangtze River basin, China, as well as the correlation of CA activity with HCO(3)(-) concentration and CO(2) sink flux. Different degrees of CA activity could be detected in almost all of the water samples from different geological eco-environments in all four seasons. The CA activity of water samples from karst areas was significantly higher than from non-karst areas (P

Sequestration of atmospheric CO2 is attracting considerable attention due to global warming. Karstification significantly affects the sequestration of atmospheric CO2. Microorganisms and carbonic anhydrase (CA) can promote karstification, but the actual contribution of microorganisms and CA to karstification intensity (KaI) under natural conditions remains unclear. In this study, the influence of soil microorganisms and CA on KaI and main regulatory factors was investigated through field soil column dissolution experiments. Guilin Yaji Karst Experimental Site was used as the typical karst area, and Silai clastic rock area was the control area. Results showed that surface soil in various geomorphological positions in the karst area presented KaI in the following order: saddle > slope > depression. Addition of composite CA-producing microbial inoculants or composite inhibitors regulated CA activity in surface soil, thereby influencing KaI. Illumina high-throughput sequencing results indicated that the soil dominant bacterial phyla were generally the same in various experimental groups and plots, and the bacterial abundance in surface soil was regulated by additives. Addition of composite microbial inoculants increased the total bacterial abundance of surface soil, whereas addition of composite inhibitors reduced the total bacterial abundance. The ratio of CA-producing bacterial abundance to total bacterial abundance was the largest at saddle in the karst area, in corresponding to the highest KaI. This finding suggested the significant role of CA-producing bacteria in promoting KaI. Correlation analysis revealed that the main soil regulatory factors of KaI were soil pH, total hydrogen, total organic carbon, and exchangeable calcium.

Schizochytrium sp. is one of the most promising marine oleaginous microorganisms for industrial production of docosahexaenoic acid (DHA). In this study, the exogenous supplementation of 1 mM sesamol to the fermentation medium effectively prevented the peroxidation of polyunsaturated fatty acids in the fermentation process, which thereby significantly increasing the lipid and DHA yield by 53.52% and 78.30%, respectively. The addition of sesamol also increased the total antioxidant capacity of cells and induce the gene expression of polyketide synthase and antioxidant enzyme system. Moreover, the supply of nicotinamide adenine dinucleotide phosphate was regulated by sesamol by inhibiting the malic enzyme activity and promoting the glucose-6-phosphate dehydrogenase activity. Finally, fed-batch fermentation showed that the addition of sesamol significantly enhanced the DHA yield by 90.76%. This study provides an important reference for enhancing the DHA productivity of Schizochytrium sp. in industrial fermentation.

Autotrophic carbon-fixing bacteria are the main drivers of carbon sequestration and elemental cycle in wetland ecosystems. Their relationship with environmental factors in karst soils such as soil organic carbon (SOC) fractions, which are affected by natural degradation and human disturbance, is key to understanding the biological mechanisms of karst wetland ecosystem deterioration and restoration. In this study, the abundances of the Calvin cycle functional genes cbbL and cbbM as well as the characteristics of carbon-fixing bacterial communities were compared in soil samples from a native wetland, naturally degraded wetland, and reclaimed farmland wetland in the Huixian karst wetland, Guilin, China. The abundances of the cbbL gene in the degraded wetland and reclaimed wetland soils varied significantly among the seasons (P winter). The influence of the season on the abundances of the cbbL and cbbM genes was more significant than that of the wetland state. The structures of carbon-fixing bacterial communities were similar in the three states of karst wetland soils, mainly consisting of Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria in the phylum Proteobacteria. However, the relative abundances of some dominant bacterial genera were significantly different. Thiomonas and Bradyrhizobium had a relatively high abundance in the native wetland soil and the degraded wetland soil, respectively, while Ferriphaselus and Sulfuricaulis were more abundant in the reclaimed farmland wetland soil. The main soil factors affecting the structure of the carbon-fixing bacterial communities were the SOC and its fractions dissolved organic carbon (DOC), microbial biomass carbon (MBC) and readily oxidizable organic carbon (ROC), as well as the soil temperature. Anthropogenic activities such as wetland transition to farmland have caused significant changes in karst soils and the characteristics of carbon-fixing bacterial community in this karst wetland soil ecosystem.

A codon optimized synthetic alkaline thermostable Thermobifida fusca β-mannanase ManB ( KJ806638 ) was expressed in Pichia pastoris and used in ramie degumming. To improve the expression level, a multi-copy secretion expression vector pAOhr was constructed to introduce the ManB gene into Pichia pastoris GS115. The highest secretion yield was obtained from a transformant strain containing six copies of ManB gene. The size of ManB protein was 34 kDa in SDS-PAGE and the secreted protein was the main protein in the culture broth. The optimal activity region of ManB was at pH 7–9 and the enzyme was quite stable at pH 6–10. At pH 9, the specific activity of ManB was 493.8 IU/mg and the optimum temperature was 70–75 °C. ManB appeared to be inhibited by Tris buffer. Molecular docking showed that Tris molecule can bind to the enzyme active site. ManB exhibited high activity for locust bean gum, whereas it showed in practice no activity for CMC-Na. Ramie degumming was performed with combined treatment by ManB and Bacillus sp. HG-28 expressing pectinase and xylanase. The obtained results demonstrated that the combination treatment with additional mannanase enzyme was more efficient in removing the gums than the treatment merely by the bacterial strain.

Atmospheric CO 2 are closely connected to climate change and global carbon cycle. Karst processes in CO 2 -H 2 O-carbonate system can absorb atmospheric CO 2 . Carbonic anhydrase (CA) can efficiently catalyze the interconversion between CO 2 and HCO 3 − , which may help accelerate karst processes. To explore the influence of bacterial CA on CO 2 capture capacity in CO 2 -H 2 O-carbonate system, a flow dissolution experimental device was designed to simulate CO 2 absorption by rainwater infiltration or surface water flushing. Results showed that, when bacterial CA was added into the experimental system, it increased not only the rate but also the quantity of CO 2 absorption, and the CO 2 captured by limestone or dolomite dissolution increased by at least 18.9% and 22.1%, respectively. However, excessively high concentration of CO 2 was not conducive to carbonate rock dissolution by bacterial CA. In accordance with the fixed carbon caused by global carbonate dissolution, the contribution of soil CA to the CO 2 sinks may reach 0.567–1.938 × 10 14 gC/a. In summary, the addition of microbial CA could increase the CO 2 capture in the form of DIC in CO 2 -H 2 O-carbonate system, thereby increasing the carbon sink potential of karst systems. The results help establish a promising approach for reducing CO 2 emission by microbial CA.

Lepidium meyenii Walpers, a well-known crop with a long history in Peru, has been utilized in the Andean region as foodstuff and medical supply. In our study, ethanol extract was obtained from the dried root of this plant and then chemically investigated. Two new imidazole alkaloids, namely, lepidiline C (1) and D (2), along with two known imidazole alkaloids (lepidiline A and B), were isolated through semi-preparative high-performance liquid chromatography (HPLC). Their structures were elucidated through HPLC⿿mass spectrometry, 1D and 2D proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet spectrophotometry.

A comparison of the glycerol and glucose effect on the fermentation of Schizochytrium sp. S056 indicated that glycerol increased the docosahexaenoic acid (DHA) concentration of the total fatty acids to 52.28% which was 31.75% higher than that produced by glucose. The underlying mechanism of the DHA content increase by the glycerol treatment, however, remains to be clarified. Transcriptome analysis was used to study the effect of the carbon source on genetic regulation and metabolism. These results revealed that when Schizochytrium sp. S056 was cultured with glycerol, the glycolysis and branched-chain amino acids (valine, leucine, and isoleucine) metabolism-associated genes were substantially up-regulated, resulting in increased overall acetyl-CoA production. Additionally, genes involved in the gluconeogenesis, pentose phosphate and citric acid cycle pathways were significantly down-regulated, resulting in more acetyl-CoA for fatty acid formation. Specifically, the expression of the fatty acid synthase (FAS) gene, which is involved in fatty acid biosynthesis, was up-regulated by the glycerol-increased production of acetyl-CoA. Accordingly, the enhanced FAS system led to a significant increase of the content of DHA. Therefore, overexpression of the FAS gene favors the accumulation of DHA. Furthermore, these results were also corroborated through the inhibition of the FAS system by cerulenin treatment.

Pyracantha fortuneana fruit is a potential source of nutraceutical and therapeutic agents. We investigated the effects of P . fortuneana fruit extract (PFE) on hyperlipidaemia, obesity, and oxidative stress in rodents. PFE significantly decreased body weight, triacylglycerol, total cholesterol, low-density lipoprotein-cholesterol, and malondialdehyde levels. By contrast, PFE elevated high-density lipoprotein-cholesterol levels, glutathione peroxidase, and superoxide dismutase activities in both preventive and therapeutic rodent groups. These results indicate that the amelioration of PFE on hyperlipidaemia and obesity was highly associated with the improvement of endogenous antioxidant activity. Polyphenols and saponins were quantified by spectrophotometric and HPLC methods. Several new saponin compounds were identified by liquid chromatography and tandem mass spectrometry as ursolic acid (UA) derivatives. The quercetin, rutin, and procyanidin treatments in HepG2 cells showed increases in low-density lipoprotein receptor activity and apolipoprotein-A1, but UA less affected these protein activities. Therefore, PFE could be promising in ameliorating chronic metabolic diseases via multiple constituent synergies.

Microalgal abundance and community composition are important aspects impacting carbon sequestration in aquatic karst ecosystems. At present the relationship between Ca2+ and HCO3− concentrations and microalgal communities specifically in aquatic karst environments is not well understood. In this study, the microalgal and bacterial community structures were investigated in the aquatic karst environments with different Ca2+ and HCO3− concentrations in a watershed of the Huixian karst wetland, Guilin, China. Community analysis was based on the use of Illumina sequencing of 23S rRNA and 16S rRNA genes, respectively. The results showed that eukaryotic microalgae were dominant at sites with high concentrations of HCO3− and Ca2+, while cyanobacteria were the major microalgae at sites with low concentrations of HCO3− and Ca2+. Cyanobium was the most dominant Cyanobacterial genus with a relatively high abundance in karst water samples. The microalgal community was affected by the concentrations of Ca2+ and HCO3−, nitrogen sources (concentrations of ammonium and nitrate especially), and bacterial diversity and community composition. Flavobacteriia and Gammaproteobacteria were the major bacterial classes associated with microalgae. The water carbonic anhydrase (CA) activity was significantly positively correlated with the relative abundance of Chlorella, which was the dominant genus in the microalgal community. The data suggest that Chlorella may play a leading role in CA activity and carbon stabilization capacity of microalgal communities in this aquatic karst ecosystem.

Degumming is the most critical step in the preparation of ramie fibers for textile application. However, the traditional chemical degumming process, which uses large amounts of alkali at high temperature, has been a severe challenge to the environment. In this study, a bacterial strain Pectobacterium carotovorum HG-49, was isolated and successfully applied to the degumming of ramie fibers at an industrial scale. The gum removal ratio can reach 82.16% within 16 h of degumming, which is more efficient than most reported methods. Analyses of changes in bacterial populations and community structures over the degumming process showed that strain HG-49 is the dominant bacteria at all time points with a maximum concentration of 2.17 × 109 CFU/mL at 12 h. The maximum activities of pectinase, mannanase, and xylanase reached 110.89, 39.89, and 32.17 U/mL at 10–12 h, respectively. The residual gum content of refined ramie fibers decreased to 1.85% after degumming, and their bundle breaking tenacity and whiteness reached 5.03 cN/dtex and 52, respectively. These values exceed the first-class standards of China textile criteria. Scanning Electron Microscopy (SEM) and Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR FT-IR) analysis further confirmed the excellent degumming effects of the proposed method. The whole genome sequencing of strain HG-49 was completed, and 28 degumming enzyme genes were found, which explains its high-efficiency degumming abilities. Moreover, because the method abandons the use of acid and greatly reduces alkali consumption, it is eco-friendly. This study provides a promising cleaner degumming method for the ramie textile industry.

The potentiality of PGPR and biocontrol bacteria in agriculture is steadily increased as it offers an attractive way to replace the use of chemical fertilizers, pesticides and other supplements. This study aimed to investigate the effects and potential mechanism of inoculation with two Bacillus velezensis strains on the growth and quality improvement of Anoectochilus roxburghii (Wall.) Lindl. (MRH) and Anoectochilus formosanus (Wall.) Lindl. (YYB). The control treatment was non-inoculated (CK) and the coculture treatments were inoculated with Bacillus velezensis D2WM (D2), Bacillus velezensis ZJ-11 (ZJ-11), Bacillus velezensis D2WM and Bacillus velezensis ZJ-11 (D2+ZJ-11). The fresh weight, plant length, amount of active compounds, as well as chlorophyll content were significantly increased under D2, ZJ-11, and D2+ZJ-11 treatments compared with MRH and YYB plants in CK. In particular, D2+ZJ-11 treatment resulted in the greatest growth promotion. In MRH and YYB plants, the fresh weight increased by 82.6 % and 106.6 %, the kinsenoside content increased by 9.33 % and 21.65 % per gram, and the flavonoid content increased by 44.70 % and 21.07 % per gram, respectively. D2WM and ZJ-11 were capable of secreting siderophore, phytase, indole-3-acetic acid (IAA), and zeatin to promote plant nutrient assimilation and growth. Moreover, when D2WM and ZJ-11 were both inoculated with Anoectochilus roxburghii, the rhizosphere soil enzyme activities of urease, phosphatase, and invertase were significantly higher than control, thus helped to provide more nutrients to Anoectochilus. Further, the analysis of microbial community diversity indicated the increase of abundance of beneficial microorganisms, such as nitrogen-fixing bacteria, Basidiomycota and Ascomycota both of which are known to fuel their plant partners with mineral nutrients from the soil. Additionally, the abundance of pathogens belonging to Xanthobacteraceae as well as Cladophialophora and Penicillium were reduced. Hence, coculture with beneficial microorganisms can improve the growth and quality of Anoectochilus plants via promoting nutrient assimilation and regulating rhizosphere microbial community. This technique is of great application value in promoting the efficient cultivation and production of high-quality plant materials.

Maca has been traditionally used to enhance sexual behavior and fertility. Recently, maca's neuroprotective effects have been reported. The purpose of this study was to investigate whether the ethanol extract of maca (EEM) (100 mg/kg/bw, 200 mg/kg/bw, 400 mg/kg/bw, p.o.) exerted neuroprotective effects in corticosterone (CORT)-induced (40 mg/kg/bw, s.c.) rats, to determine the neuroprotective effects of EEM (12.5, 25, 50 μg/ml) and macamides in H2O2-induced (50 μM) PC12 cells. The acute toxicity (2000 mg/kg/bw, p.o.) and subacute toxicity (200 mg/kg/bw, 500 mg/kg/bw, 1000 mg/kg/bw, p.o.) of EEM were evaluated by mouse models. EEM reversed CORT-induced abnormal behaviors, reduced the contents of TNF-α, IL-6 in hippocampi, and increased the positive cells of doublecortin (DCX), bromodeoxyuridine (BrdU) and DCX + BrdU in the hippocampus of rats. Moreover, EEM and 4 macamides remarkably increased the cell viability in H2O2-induced PC12 cells. EEM promoted the phosphorylation of IκBα and p65, suppressed the NF-κB activation, and inhibited the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and their mRNA levels in H2O2-induced PC12 cells. In conclusion, EEM could exert neuroprotective effects in CORT-induced rats and in H2O2-induced PC12 cells. Moreover, EEM did not present relevant toxicity after exposure to single and repeated doses.

High-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) is an important technique for amino acid analysis. As a common derivatization reagent, phenyl isothiocyanate (PITC) is widely used in this field. However, researchers have long faced the critical problem of coupling efficient separation with simultaneous, sensitive MS detection. In this work, a novel HPLC–ESI-MS method based on PITC derivatization was established for detecting amino acids. Complete separation of 15 amino acid derivatives was achieved with an Agilent Eclipse plus C18 column maintained at 30 °C in 29 min. The mobile phase was 0.05% formic acid aqueous solution (A) and 70:30 (v/v) acetonitrile–water (B). The spectral data of PITC-amino acids are indicative of losses of 17 Da (NH3), 18 Da (H2O) and/or 46 Da (CO + H2O) from the parent protonated molecules. Assessment of this method revealed that response curves were linear within the range of 630–20,156 μmol L−1. LOD and LOQ ranged within 111–816 and 311–2721 pmol μL−1, respectively. The method was validated with precision values of 0.924–6.578% (intraday, n = 6) and 3.327–8.962% (interday, n = 6 in 3 d). Therefore, the proposed method is a simple, robust alternative to current methods of detecting amino acids.

The industrial production of diosgenin in China generates a large amount of high-sugar wastes with low bioavailability, which causes serious pollution to the environment. In this study, a new clean and efficient process for the production of diosgenin was developed using sugars through in situ high-valued transformation. The sugar mixture from Dioscorea zingiberensis C.H. Wright contained abundant beneficial components. Nine typical microorganisms that produced intracellular products were evaluated. Saccharopolyspora spinosa was selected for recursive protoplast fusion to increase the spinosad yield by 46.3% compared with that of the wildtype. Diosgenin and spinosad co-production was conducted in a 100L bioreactor, with pH controlled by adding glucose. The biological oxygen demand of the effluent water decreased from 15,000mg/L to 450mg/L; hence, the proposed process is environment friendly.

Maca (Lepidium meyenii Walp.) is a traditional medicine and nutritional supplement from South America with many pharmacological effects, such as enhancing female and male fertility, improving sexual dysfunction, preventing osteoporosis and relieving menopausal syndrome. Until now, the active principles behind most of these effects have not been clarified, severely hindering the exploitation and application of maca products. In this study, the effective anti-osteoporotic components of maca are uncovered. Through virtual screening against estrogen receptor and verification of pharmacological activity on osteoblasts, we found that N-benzyl-palmitamide is an active constituent of maca in preventing osteoporosis. As well, N-benzyl-palmitamide promotes osteoblast proliferation, differentiation and mineralization, plus it benefits bone formation through enhanced expression of osteogenesis-related genes, such as bone morphogenetic protein-2, core binding factor alpha 1, type 1 collagen and alkaline phosphatase. The effects of N-benzyl-palmitamide on enhanced bone formation are very likely through the estrogen receptor pathway, as N-benzyl-palmitamide also increases the expression of ERα and ERβ genes. Clarifying single-component biological activity will greatly enable exploitation and application of maca as a medication and health supplement to prevent osteoporosis.

With the growing energy crisis and environmental pollution worldwide, the environmental benefits of biodiesel have made it a more attractive product. However, the production of biodiesel feedstock can only satisfy a small fraction of the demand for transportation fuels, and the cost of this feedstock is the main obstacle to the commercialization of biodiesel. The marine microalgae Schizochytrium is a promising producer of biodiesel feedstock because of its short life cycle, high cell density, high lipid content and high palmitic acid (PA) content, all of which can improve the quality of biodiesel. In this study, a sequential optimization of the culture medium, based on response surface methodology (RSM), was employed to increase the PA production in Schizochytrium and lower the process cost. As a result, the PA production by Schizochytrium sp. S056 increased from 14.69 ± 0.62 g/L to 25.95 ± 0.11 g/L using the optimal conditions. In addition, the quality analysis of the crude lipid showed that the lipid produced by the microbial process is a promising feedstock for biodiesel production.

Industrial production of ramie fibers by chemical degumming results in problems such as environmental pollution and inferior fiber quality, which are unsustainable and hinder the development of relevant industries. For these reasons, an in-situ microbial degumming process with direct application of Bacillus sp. HG-28 was developed and presented here to solve these problems. This strain was selected for its high degumming ability and low cellulose damage. In the degumming process, the gum in ramie bast fibers induced the strain to secrete high activities of pectinase and xylanase, both of which were proven to be essential for degumming. After 16 h of degumming, gum content reduction was measured to be 76.92% while cellulose loss could not be evidently detected. This is more efficient than other reported microbial degumming methods that do not use microorganisms directly. According to the results of in-situ microbial degumming in industrial scale, the residual gum content of degummed ramie fibers decreased to 1.81%, the bundle breaking tenacity reached 5.09 cN/dtex and, additionally, the consumption of chemicals, water and energy were significantly reduced, proving superior to chemical process. This study provides a sustainable alternative for the conventional chemical degumming industry.

Sucrose metabolism generates material and energy foundations and regulates the biosynthesis of secondary metabolites in plant cell cultures. However, this regulation mechanism is unclear. The neutral/alkaline invertase (NINV) gene is among the key enzymes in sucrose hydrolysis. Thus, the complete open reading frame (ORF) of this gene was obtained from Taxus chinensis cells. This gene was also successfully expressed in Escherichia coli . The expressed protein was verified through the function of sucrose cleavage. Furthermore, the transcriptional levels of the NINV gene can respond to the sucrose concentration changes and relate to taxane biosynthesis in T. chinensis cells. Subsequently, we developed the gene expression vector and introduced it into T. chinensis suspension cells to confirm the role of the NINV gene in taxane biosynthesis. Results indicated that this gene was overexpressed and that it significantly enhanced the expression of the taxadiene synthase (TAS) gene, as well as the biosynthesis of the seven detected individual taxanes. Thus, NINV-mediated sucrose metabolism can promote the biosynthesis of secondary metabolites. This study presents new concepts and approaches to increase the production of secondary metabolites in plant cell cultures.

Proanthocyanidins (PCs) with poor bioavailability were argued for their health benefits. In this study, water-soluble polymeric polyphenolic PCs fractions from Pyracanthafortuneana fruit were used to investigate whether the presence of PCs is correlated with the increased cell antioxidant activities (CAA) of quercetin (Q). The results indicated that the most decrement in the values of EC50, which Q inhibited peroxyl radical-induced DCFH oxidation effective in the HepG2 cells, was observed to be 2.91 (vs. control 5.97) in the present of the fraction with 15.8 of the average degree of polymerisation of PCs (ADP). Also, the order of efficacy was the same with the ADP of PCs. Further, this effect is associated with the improvement of the solubility and stability of Q after the addition of the PCs. Our current study suggests that the additive effects of PCs on small molecular polyphenols may be responsible for their antioxidant benefits in vivo.

A novel method utilizing microbial treatment for cleaner production of diosgenin from Dioscorea zingiberensis C.H. Wright (DZW) was presented. A new Bacillus pumilus HR19, which has the great ability to secrete pectinase, was screened and applied in the microbial treatment. Low-pressure steam expansion pretreatment (LSEP) was employed in advance to assist microbial treatment efficiently in releasing saponins, which are the precursors of diosgenin. Compared with the traditional process of acid hydrolysis, this novel process reduced the consumptions of water, acid and organic solvent by more than 92.5%, 97.0%, 97.0%, respectively, while simultaneously increasing the diosgenin yield by 6.21%. In addition, the microbial treatment was more efficient than enzymatic treatment, which arised from that microorganisms could be induced to secrete related enzymes by the compositions of DZW and relieve product inhibition by utilizing enzyme hydrolysates.

One of the most promising biological sequestration technologies of CO 2 is the enzyme catalyzed CO 2 sequestration into stable and environmentally friendly mineral carbonates. The present manuscript focused on the biocatalytic precipitation of CaCO 3 by extracellular carbonic anhydrase (CA) which was extracted and purified from the culture of Bacillus cereus . The kinetics of CaCO 3 precipitation catalyzed by the bacterial CA in the presence of different enzyme concentrations was investigated through the gaseous diffusion system. The polymorph and morphology of CaCO 3 crystals obtained in the precipitation process were also analyzed using XRD, FTIR and FESEM. The results showed that the change in the amount of deposited Ca 2+ during the process of CaCO 3 precipitation catalyzed by bacterial CA of different concentrations was well fitted with the exponential model. The enzyme concentrations of 0.2–2.0 U/mL were beneficial to CaCO 3 precipitation, however, overhigh enzyme concentration (8.0 U/mL) was not conducive to CaCO 3 precipitation. The integrated results of XRD, FTIR and FESEM analysis showed that there were significant differences in the morphologies of CaCO 3 crystals among different enzyme concentrations. Vaterite was present at the lower concentration of CA, and the higher concentration of CA favored the formation of calcite. Therefore, different polymorphs and morphologies of CaCO 3 crystals can be produced in the presence of different concentrations of CA. Furthermore, the role of bacterial CA in CaCO 3 precipitation was related to the electrostatic adsorption of Ca 2+ on CA enzyme protein and the preferential adsorption of CA enzyme protein on the crystal faces, in addition to the enzymatic catalysis.

Yields of paclitaxel decreased with repeated subculturing of Taxus media cells. We used minimal growth conservation and manipulation of genome methylation to sustain paclitaxel production by Taxus media cell cultures. The subculture period of Taxus cells can be prolonged to 180 d by incubating them at a low temperature (5 °C). Paclitaxel levels increased in the cells after conservation and during the first recovery subculture cycle, and then decreased during the subsequent recovery subculture cycle. Analysis of genetic variations in these cultures using amplified fragment-length polymorphism (AFLP) technology identified only two polymorphic bands associated with the second and sixth recovery cycle cultures. However, the results of high-performance liquid chromatography indicated that DNA methylation increased during the course of repeated subculturing. A decrease in DNA methylation level caused by treatment with 5-Aza-2′-deoxycytidine coincided with an increase in paclitaxel levels. Simultaneous exposure to both methyl jasmonate and 5-Aza-2′-deoxycytidine increased paclitaxel levels to 320.43 μg g −1 (dry weight), which is more than six times the paclitaxel content before conservation. To our knowledge, this is the first report about improving paclitaxel production by ensuring sustainable use of Taxus cells.

Biogenic precipitation of calcium carbonate (CaCO3) has attracted much attention due to its role in many geological processes, applications of Geological and Civil Engineering as well as environmental treatments. The present paper focused on the biocatalytic precipitation of CaCO3 by the extracellular carbonic anhydrase (CA) extracted and partially purified from the culture of Bacillus cereus. The kinetics of CaCO3 precipitation catalyzed by the bacterial CA at different initial concentrations of Ca2+ (C0(Ca2+)) was investigated through the gaseous diffusion system. The polymorph and morphology of CaCO3 crystals obtained in the precipitation process were also analyzed using XRD, FTIR and FESEM. The results showed that in the process of CaCO3 precipitation catalyzed by bacterial CA, the change in the amount of deposited Ca2+ at different C0(Ca2+) fitted well with the exponential model. Greater fluctuation of pH occurred in the water control group during the rising process of pH, while in the CA group the pH increased more steadily. This may be related to the role of CA in pH regulation. The precipitation rate of CaCO3 increased with the increasing C0(Ca2+), but overhigh C0(Ca2+) of 100 mmol/L had a certain negative influence on CaCO3 precipitation catalyzed by bacterial CA. The integrated results of XRD, FTIR and FESEM analysis showed that the C0(Ca2+) had greater effect on the polymorph and morphology of CaCO3 crystals formed in the presence of bacterial CA. The lower C0(Ca2+) favored the formation of vaterite and the higher C0(Ca2+) favored the formation of calcite.

For a better understanding of the simulation, optimization, and process control in cell cultures, good kinetic models are necessary for large scale plant cell culture. In this paper, the systematic kinetics of taxol production by Taxus media cell suspension cultures in a stirred 15-L bioreactor under substrate-sufficient conditions and the absence of inducer intervention were studied. A kinetic model of cell growth was established by logistic equation, and kinetic unstructured models of substrate consumption, product synthesis and rheological behavior were constituted, which incorporated energy spilling. These models were verified by comparing the simulation results with those obtained experimentally. These results showed that energy spilling was a key factor that must be considered in constructing unstructured kinetic models of Taxus media cell suspension cultures in a stirred bioreactor under substrate-sufficient conditions. Besides, an optimized operation measure of decreasing energy spilling was proposed. An increase of 17.64% in cell biomass and 14.88% in taxol concentration were obtained when the strategy of limiting added carbon several times was experimentally implemented in a 15-L bioreactor. Results demonstrated that these established models should be helpful in the process prediction and operation optimization to guide the production and amplification of Taxus media cell suspension cultures in a bioreactor.

The dynamics of calcium carbonate (CaCO(3)) precipitation induced by microbial intracellular or extracellular carbonic anhydrase (CA) at initial pH 6.0, 6.5, 7.0 and 8.0 were investigated through the gaseous diffusion method. The results indicated that both the intracellular and extracellular CA could promote CaCO(3) precipitation. The Ca(2+) ions in the enzymatic systems at initial pH 8.0 were completely deposited at 48 h, which were respectively 21 h, 15 h and 14 h earlier compared with that at initial pH 6.0, pH 6.5 and pH 7.0, indicating that higher pH favored CaCO(3) precipitation in the experimental pH range, and was beneficial to the catalytic action of microbial CA on CaCO(3) precipitation. In addition, XRD analysis indicated that the CaCO(3) precipitates were mainly calcite crystals in the presence of microbial CA. With increasing deposition time, the crystals gradually changed from prism shape to pyramid-like or irregular polyhedral shape based on FESEM analysis.

Traditional batch fermentation leads to a higher energy consumption and lower production capability because of longer culture time. In this work, a pilot scale bioreactor composed of a 3000 L fermentor and external ceramic microfiltration equipment was used to perform cell-recycle fermentation. Repeat feeding medium was also used to relieve the substrate inhibition. In such pilot system, the maximum yield and productivity of l (+)-lactic acid production reached 157.22 ± 3.42 g/L and 8.77 ± 0.15 g/L/h which were 4.23% and 315.64% higher than those of batch fermentation, respectively, when equal amount of sugar was consumed. The cost of l (+)-lactic acid production was successfully reduced about two-thirds by the increase of yield and productivity. 12 rounds of cell-recycle fermentations were successfully achieved in the pilot system. The membrane filtration productivity reached to 61.27 ± 2.74 L/m 2 /h which increased 172.80%, while the cell damaging rate dropped to 3.88 ± 0.18% which decreased 85.77%, compared with those of the ultrafiltration. Furthermore, the ceramic microfiltration membrane showed advantages in tolerance for the temperature, pressure and acid, compared with the organic ultrafiltration membrane. The experimental results indicated that the method could give a reference for low cost production of l (+)-lactic acid in an industrial scale.

Eutrophication in lakes and reservoirs occurs all over the world and has attracted wide attention in recent years. MIKE21, a numerical model focusing on eutrophication in lakes and reservoirs, was used to construct a two-dimensional eutrophication model. Douhe Reservoir in North China was used for the study. A hypothetical case study was applied in order to simulate a situation in which variables like air temperature, transferred water volume, or exogenous pollutant load might reach extreme values. A “Comprehensive nutrition state index” was used to evaluate the reservoir eutrophication level in different scenarios. The causes and trend of eutrophication were found, which could be a reference for eutrophication prevention and control. The main conclusions are that: (1) the warm-water emission from the power plant is the main reason for the rise in water temperature in Douhe Reservoir, which has a not negligible effect on eutrophication; (2) changes in outside temperature and reservoir storage capacity have just a small impact on a eutrophication trend; an increase in exogenous load can rapidly escalate eutrophication in Douhe Reservoir; (3) higher water temperature and higher TN/TP ratio lead to a lower Chlorophyll a concentration in water bodies of a nearby warm-water discharging spot than in the other two monitored areas.

The Bi 5− x La x Ti 3 Co 0.5 Fe 0.5 O 15 (0 ≤ x ≤ 0.4) ceramics were successfully synthesized by a modified Pechini process. The samples were characterized by X-ray diffraction and no impurity phase has been detected. The cell volume of the composites increases monotonously with the increase of La content, which indicates that La ions have been incorporated into the lattice of Bi 5 Ti 3 Co 0.5 Fe 0.5 O 15 . The magnetic measurements show that La doping on Bi sites has enhanced the magnetization of Bi 5− x La x Ti 3 Co 0.5 Fe 0.5 O 15 (0 ≤ x ≤ 0.4). Both the dielectric constants and loss tangent of all the samples decrease on increasing frequency and then become almost constant at room temperature. The La doped Bi 5 Ti 3 Co 0.5 Fe 0.5 O 15 samples exhibit improved dielectric and ferroelectric properties, with higher dielectric constant enhanced remnant polarization and lower losses at room temperature.

The effect of microbial carbonic anhydrase (CA) on the calcium carbonate (CaCO3) precipitation was systematically investigated comparing to other biological factors (bovine CA, bovine serum albumin, carboxymethyl chitosan and glutamic acid). The results showed that the precipitation rate of Ca2+ in the presence of either microbial CA or bovine CA was faster than that in the presence of 1% bovine serum albumin, 1% carboxymethyl chitosan or 1% glutamic acid, respectively. In addition, XRD analysis indicated that the dominant CaCO3 crystal phase was calcite. The CaCO3 crystal morphologies mainly showed cubic and polyhedral shapes induced by microbial CA, and became multiformity induced by other factors from FE-SEM analysis. These results suggested a novel approach for biomimetic synthesis of CaCO3 materials by microbial CA.

A headspace solid-phase microextraction (HS-SPME) method was used to extract the essential oil of Osmanthus fragrans Lour., which was then analysed by gas chromatography–mass spectrometry (GC–MS) at four different stages of flowering. The primary chemical components of the essential oil extracted from O. fragrans were linalool and its oxide, α-ionone, β-ionone, nerol, γ-decalactone, 9,12,15-octadecatrienoic acid, and hexadecanoic acid, most of which were at their highest concentrations in extracts obtained from flowers at the initial flowering stage. HS-SPME using a PDMS/DVB fibre is a simple, rapid, and solvent-free method for the extraction of volatile compounds emitted from living O. fragrans. HS-SPME with GC–MS can be used to determine the aromatic maturity in O. fragrans, to decide the optimal harvest date. The initial flowering stage was the best time to harvest O. fragrans.

The root of Tripterygium wilfordii (TWH) is a traditional Chinese herb used to treat the immune-related diseases such as rheumatoid arthritis, whereas the leaf and twig of TWH was considered useless and discarded. We performed a study on the anti-inflammatory effects on the leaf and twig portion agent using carrageenan- and adjuvant-induced paw edema in rats. They showed a marked inhibitory effect on edema in both models of inflammation in rats, at the dose of 50, 100 and 200 mg/kg, especially on secondary immunological arthritis. Based on this study, we confirmed that the leaf and twig of TWH is a potentially useful drug suitable for further evaluation for rheumatoid arthritis and can replace root of TWH.

Comprehensive utilization of the mixture of oil sediments (OS) and soapstock (SS) for producing FAME and phosphatides was investigated. A process consisting of three steps was employed for obtaining high conversion and by-product. In the first step, the OS–SS mixture was extracted with ethyl ether and the mixture was divided into three phases. The organic top phase contained triglycerides and phosphatides was extracted with cooled acetone and the acetone insoluble (phosphatides) was obtained. At the same time, triglycerides were separated also. In the second step, soap phase was then acidified with sulfuric acid to yield fatty acid. This “high-acid” acid oil was efficiently converted to methyl esters by acid-catalyzed esterification. The esterification reaction has been carried out with 5:1 methanol/oil (mol/mol) in the presence 3% H 2 S0 4 (wt.%) as an acid catalyst at 85 °C for 5 h. FAME recovery under these conditions was 92.1% of theoretical. In the third step, alkaline catalyzed transesterification process converts the triglycerides to its mono-esters and glycerol. The optimized variables, 6:1 methanol/oil (mol/mol) with 1% NaOH (wt.%) reacted at 65 °C for 1 h, giving a maximum ester yield of 94%. Five important fuel properties of FAME from the OS–SS mixture were found to be comparable to those of No. 2 diesel fuel and conforming to both the American and German standards for biodiesel.

Green teas (Camellia sinensis) from the non-Karst and the Karst areas of Yichang, Hubei Province, PR China, have been compared as regards l-proline content of the green tea and the quality, as well as the soil properties. The results showed that some qualities of green tea from the Karst region, such as a higher content of l-proline and a lower contents of aluminum and fluoride were beneficial. Other qualities of green tea from the Karst region, for example, the lower content of tea polyphenols, were not beneficial. The differences in amino acids and caffeine contents were not statistically significant.

Arachidonic acid is an essential fatty acid in human nutrition. Improvement of arachidonic acid production by fungus Mortierella alpina was investigated. The time courses of fungal biomass, lipid and arachidonic acid production with different initial glucose concentrations were examined to study dynamic characteristics of arachidonic acid production on a shaker flask scale. Results showed that low initial concentrations of glucose were good for fungal growth at the early days of cultivation. When residual glucose concentration was under 10 g/L, fungi stopped growing because of inefficient energy source and carbon source needed for their growth. Meanwhile, the lipids were still accumulated for a period even though fungi stop growing. A great increase of arachidonic acid content in lipids occurred after the glucose was exhausted. To obtain high arachidonic acid yield, a fed-batch process was developed according to the dynamic analysis. Glucose and nitrate were included in the feed medium. Low initial glucose concentration (50 g/L) and nitrate concentration (3 g/L) were used to shorten the lag phase of fungal growth. Twenty grams per liter per day glucose and 1.5 g/(L day) nitrate were fed to the medium at the 3rd, 4th and 5th days of cultivation. A good arachidonic acid yield (7.74 g/L) on the 8th day was obtained, which was a great improvement over that of batch cultures.

Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina M-23 were examined to determine their effects on the growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous. The results showed that different fungal elicitor could cause diversely stimulating effects. Among the fungal elicitors tested, the M. mucedo elicitor concentration of 30 mg l(-1) promoted the biomass and total carotenoids yield most remarkably, resulting in 69.81+/-6.00% and 78.87+/-4.15% higher than the control, respectively. At the concentration of 30 mg l(-1), R. glutinis elicitor stimulated the highest astaxanthin yield with a 90.60+/-5.98% increase compared to the control. The R. rubra elicitor concentration of 30 mg l(-1) resulted in the optimal total carotenoids and astaxanthin content to be 42.24+/-0.49% and 69.02+/-0.72% higher than the control, respectively. At the concentration of 30 mg l(-1), R. rubra elicitor gave the highest increase in the ratio of astaxanthin in total carotenoids by 18.85+/-0.11% of the control.

With multimedia developing rapidly, copyright protection and authentication is more and more important. Digital watermark is one of the most efficient methods to protect multimedia products. Print and scan attack is a challenging problem for most of digital watermarks. In this paper, the geometric distortion and halftone in print-and-scan process is analyzed for the sake of presenting a model of print-and-scan. A digital watermarking countermeasure is proposed. Experiment results verify the efficiency of the proposed watermarking countermeasure. The proposed work is promising in application of copyright protection and anti-forgery for paper media.

In natural karst systems, limestone diagenesis can be significantly influenced by bacterial activity in the soil horizon. Here, we investigate the effects of microorganisms on the elements migration of calcium and magnesium in karst soil systems by using different microbial treatments in simulated soil-limestone systems. Two bacterial strains, GLRT102Ca and JFSRT303 were specially studied. The leaching and release of Ca2+ in the experiments was characterized by a rapid initial increase followed by a sharp decrease before a gradual approach to equilibrium. In contrast, the Mg2+ concentrations in the leachates showed an initial decrease before a gradual approach to equilibrium. Microorganisms significantly promoted Ca2+ and Mg2+ migration in the simulated systems. The total amounts of Ca2+ and Mg2+ in leachates varied with microbial treatments. The soil + GLRT102Ca columns showed the highest total amount of Ca2+ in leachates. This increased by a factor of 2.2 relative to the control columns. The highest total amount of Mg2+ in leachates was presented in the soil + JFSRT303 columns, which leached 58.0% more total amounts of Mg2+ than the control columns. The activities of a microbial specific enzyme, carbonic anhydrase (CA), present in the investigated columns were also examined. Varying levels of CA activities were detected in the leachates collected from soil columns with microbial activity. This suggests that the microbes in soil columns produced and released CA. The mean activity of CA in leachates was significantly correlated with total amount of Ca2+ in leachates (r = 0.86, P

The role of lipoxygenase (LOX) in the elicitor-induced taxol production of Taxus chinensis cell cultures was studied. Fungal elicitor F 5 treatment effectively increased LOX activity and taxol production. The LOX activity inhibitor phenidone could inhibit induced LOX activity and taxol production, suggesting that LOX pathway might be involved in elicitor-induced taxol production. Methyl jasmonate (MJ) also activated LOX activity and taxol production. Phenidone could inhibit LOX activity and taxol production challenged by MJ, suggesting that exogenous MJ may induce taxol production via activating the intracellular LOX pathway. For better understanding the role of LOX in T. chinensis , some biochemical features of LOX were also investigated. Results showed that at least 49.18% LOX was associated with membrane and the molecular weight of LOX was 93 kDa.

To determine the role of microbes in karst processes, it is necessary to investigate the ecological distribution and characteristics of soil microorganisms in karst areas. In this paper, a preliminary study was carried out in two different karst areas of Southwest China: Nongla and the Guilin Yaji Karst Experimental Site (Guangxi). Soil samples from 10–20 cm in depth were analyzed for the number of bacteria, and the predominant bacteria were identified. Analysis showed that the amount of soil bacteria correlated highly with characteristics of the karst ecosystems, including their different geochemical environments and vegetation. The predominance of Azotobacteraceae colonies showed that the soil fertility of both types of karst areas may be improving. Also, the origin of carbonic anhydrase (CA), which could accelerate karst processes, was explored. The CA-producing bacteria were screened, and activities of extracellular and intracellular CA were measured. Obvious differences existed in intracellular and extracellular CA activities of soil bacteria between the two karst ecosystems with different vegetation conditions. This suggests that the activity of CA from soil bacteria in the two different karst areas was also correlated with karst ecosystem characteristics, including their different geochemical environments and vegetation features.

Elicitation was an efficient method for enhancing taxol production in asynchronous cultures of Taxus chinensis cells but high production of taxol was unstable in 250 ml Erlenmeyer flask and a 10 l working volume stirred bioreactor. This situation could be remedied using two synchronization methods, double phosphate starvation and low temperature treatment. Low temperature treatment could not only maintain stable high taxol production, but also further enhance taxol production compared with elicited asynchronous cultures. This resulted in the greatest taxol production of 27 mg l−1, being about 2 and 11 times higher than in asynchronous cultures and in elicited asynchronous cultures, respectively. Taxol production in a stirred bioreactor was less than in flasks, but changes of taxol production in asynchronous cultures and synchronous cultures in a stirred bioreactor were similar to those in 250 ml flask. Therefore, high stable taxol production in elicited synchronous cultures of T. chinensis cells was amenable to scaleup. The possible relationship between stable production of taxol and cell cycle phase is discussed.

Effects of three treatments of 50 mg l −1 fungal elicitors (F 5 ), 50 mg l −1 salicylic acid (SA) and 50 mg l −1 F 5 +50 mg l −1 SA were studied on membrane-lipid peroxidation. Peroxidase (POD), glucose-6-phosphate dehydrogenase (G6PDH) and taxol production in cell suspension cultures of Taxus chinensis were investigated. The results showed membrane-lipid peroxidation caused by fungal elicitor F 5 , prepared from fungus isolated the inner bark of T. chinensis , was decreased by the addition of salicylic acid, even if the latter also induced cell membrane-lipid peroxidation. F 5 +SA resulted in improving the activity of POD and G6PDH compared to single F 5 treatment, and achieved the greatest taxol production of 11.5 mg l −1 , being 1.5, 2.3 and 7.5 times higher than that of F 5 , SA and the control. By analysis of membrane-lipid peroxidation, POD and G6PDH activity, the reason combined treatment F 5 and SA improved taxol production was reduction of membrane-lipid peroxidation induced by F 5 . The studies showed that treatment of F 5 +SA on T. chinensis cultures not only gained more biomass than single F 5 treatment, but also achieved the highest taxol production.