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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.

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.

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.

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.

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.

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.

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.

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.