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2-Phenylethanol is a valuable flavoring agent with many applications. Although the bioproduction of 2-phenylethanol has been achieved by microbial fermentation, the low titer and high cost hinder its industrial-scale production. The goal of this study is to develop an efficient process for high-level production of 2-phenylethanol from L-phenylalanine. Firstly, candidate hosts for 2-phenylethanol synthesis were screened by evaluating their tolerance to 2-phenylethanol, and Bacillus licheniformis DW2 was proven to be a promising strain for 2-phenylethanol production. Subsequently, phenylpyruvate decarboxylase and alcohol dehydrogenase from different hosts were screened, and the combination of KivD from Lactococcus lactis and YqhD from Escherichia coli owned the best performance on 2-phenylethanol synthesis, and the attained strain DE4 produced 3.04 g/L 2-phenylethanol from 5.00 g/L L-phenylalanine using glucose as carbon source. Furthermore, the fermentation process was optimized using molasses as carbon source, and 2-phenylethanol titer was increased to 4.41 g/L. In fed-batch fermentation, the maximum 2-phenylethanol titer reached 5.16 g/L, with a yield of 0.65 g/g on L-phenylalanine and productivity of 0.12 g/(L.h), which was the highest 2-phenylethnol titer reported to date when molasses was used as carbon source. Collectively, this study develops a robust strain as well as the cost-efficient process for 2-phenylethanol production, which lays a substantial foundation for industrial production of 2-phenylethanol. Key points •Bacillus licheniformis is an excellent 2-PE stress-tolerant strain. •Coexpressed kivD and yqhD is most suitable for 2-PE production in B. licheniformis. •High-level production of 2-PE (5.16 g/L) was obtained by engineered strain DE4. [ABSTRACT FROM AUTHOR]

Zizyphi Spinosae Semen (ZSS) is an herbal medicine with a variety of biological activities. In this study, the oil fraction of ZSS was extracted using an ultrasonic assisted extraction (UAE) method and the value of this underutilized byproduct was explored. The effects of operational parameters of the UAE process (extraction temperature, extraction time, liquid-to-solid ration, and ultrasonic power) on the extraction efficiency were evaluated based on the yield of vitamin E and the squalene, two important compounds in ZSS oil with antioxidant bioactivities. A central composite design was used to optimize these operational parameters. The optimized UAE process resulted in a vitamin E yield of 29.58 ± 0.19 mg/100 g ZSS and a squalene yield of 820.49 ± 3.66 µg/100 g ZSS, which were much higher than those obtained through the conventional heating reflux extraction (HRE) (i.e., vitamin E yield of 18.67 mg/100 g ZSS, and squalene yield of 627.25 µg/100 g ZSS). The ZSS oil contained oleic acid and linoleic acid as the main fatty acid. In addition, the UAE-derived ZSS oil exhibited stronger activities of scavenging ABTs, superoxide anions and hydroxyl radicals, compared to the ZSS oil extracted from HRE. Collectively, this work demonstrated that UAE was an effective method to extract ZSS oil with high yield. The ZSS oil can be used as a potential an antioxidant agent in food and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

A simple high performance liquid chromatography (HPLC) method was established for identification and quantification of nine polyphenols (gallic acid, chlorogenic acid, caffeic acid, myricetrin, hyperoside, luteoloside, quercitrin-3-rhamnoside, quercetin and kaempferol) in Polygoni Avicularis Herba. The analysis was performed on a Pinnacle II C18 column (250 × 4.6 mm, 5.0 µm) with a gradient elution of an aqueous mobile phase (containing 0.3% acetic acid) modified by acetonitrile. Diode-array detector (DAD) was used to detect the analytes at four wavelengths (270, 326, 355, and 370 nm). The established method was validated with good linearity, precision, accuracy and reproducibility to determine the polyphenols in Polygoni Avicularis Herba. The applicability of this analytical method was confirmed by successful analysis of the herb samples. The results indicated that the contents of these polyphenols varied with different locations of the samples. The anti-oxidation activities of Polygoni Avicularis Herba were further evaluated based on DPPH, ABTS and superoxide anion free radical scavenging assay. The herb exhibited obvious radical scavenging capacity, and gallic acid, chlorogenic acid, myricetrin and hyperoside played important roles in this activity. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis WX-02 is a well-studied strain to produce poly-γ-glutamic acid (γ-PGA) with numerous applications. This study is to improve WX-02 strain's capability of assimilating glycerol, a major byproduct of biofuels industries, through metabolic manipulation. Through gene knockout, the GlpK pathway was identified as the sole functional glycerol catabolism pathway, while the DhaK pathway was inactive for this strain under either aerobic or anaerobic conditions. The enhancement of glycerol utilization was attempted by substituting the native glpFK promoter with the constitutive promoter (P43), ytzE promoter (PytzE), and bacABC operon promoter (PbacA), respectively. The glycerol consumptions of the corresponding mutant strains WX02-P43glpFK, WX02-PytzEglpFK, and WX02-PbacAglpFK were 30.9, 26.42, and 18.8% higher than that of the WX-02 strain, respectively. The γ-PGA concentrations produced by the three mutant strains were 33.71, 23.39, and 30.05% higher than that of WX-02 strain, respectively. When biodiesel-derived crude glycerol was used as the carbon source, the mutant WX02-P43glpFK produced 16.63 g L of γ-PGA, with a productivity of 0.35 g L h. Collectively, this study demonstrated that glycerol can be used as an effective substrate for producing γ-PGA by metabolic engineering B. licheniformis strains. [ABSTRACT FROM AUTHOR]

Acetic acid derived from fast pyrolysis of lignocellulosic biomass is a promising substrate for microalgae fermentation for producing lipid-rich biomass. However, crude pyrolytic acetic acid solution contains various toxic compounds inhibiting algal growth. It was hypothesized that such an inhibition was mainly due to the cell membrane damage. In this work, the cell membrane property of algal cells was evaluated at various conditions to elucidate the mechanisms of inhibition caused by the pyrolytic substrate solution. It was found that acetic acid itself served a carbon source for boosting algal cell growth but also caused cell membrane leakage. The acetic acid concentration for highest cell density was 4 g/L. Over-liming treatment of crude pyrolytic acetic acid increased the algal growth with a concurrent reduction of cell membrane leakage. Directed evolution of algal strain enhanced cell membrane integrity and thus increased its tolerance to the toxicity of the crude substrate. Statistical analysis shows that there was a significant correlation between the cell growth performance and the cell membrane integrity (leakage) but not membrane fluidity. The addition of cyto-protectants such as Pluronic F68 and Pluronic F127 enhanced the cell membrane integrity and thus, resulted in enhanced cell growth. The transmission electron microscopy (TEM) of algal cells visually confirmed the cell membrane damage as the mechanism of the pyrolytic substrate inhibition. Collectively, this work indicates that the cell membrane is one major reason for the toxicity of pyrolytic acetic acid when being used for algal culture. To better use this pyrolytic substrate, cell membrane of the microorganism needs to be strengthened through either strain improvement or addition of membrane protectant reagents. [ABSTRACT FROM AUTHOR]

Biofilm-based algal cultivation has received increased attention as a potential platform for algal production and other applications such as wastewater treatment. Algal biofilm cultivation systems represent an alternative to the suspension-based systems that have yet to become economically viable. One major advantage of algal biofilm systems is that algae can be simply harvested through scraping and thus avoid the expensive harvesting procedures used in suspension-based harvesting such as flocculation and centrifugation. In recent years, an assortment of algal biofilm systems have been developed with various design configurations and biomass production capacities. This review summarizes the state of the art of different algal biofilm systems in terms of their design and operation. Perspectives for future research needs are also discussed to provide guidance for further development of these unique cultivation systems. [ABSTRACT FROM AUTHOR]

Lichenysin is a biodegradable surfactant with huge potential for recovering crude oil from the oil reservoir. The current production of lichenysin is made through fermentation from wild strain of Bacillus licheniformis, which is limited by low yield. The aim of this work was to improve lichenysin-producing capability of a wide strain B. licheniformis WX-02. Lichenysin produced from WX-02 was first extracted, purified, and identified. Through the substitution of the promoter of lichenysin biosynthesis operon, the mutants B. licheniformis WX02-P43lch, WX02-Pxyllch, and WX02-Psrflch were constructed with the constitutive promoter (P43), the xylose-inducible promoter (P), and the surfactin operon promoter (P), respectively. A consistent change trend was observed between lichenysin production and lchAA gene transcription, confirming the strength of the promoters as an important factor for lichenysin synthesis. Among the three mutants, WX02-Psrflch produced the highest lichenysin yield. The production by the mutant WX02-Psrflch was further improved with the optimization of the major medium components including glucose, NHNO, and NaHPO/KHPO. Under 30 g/L glucose, 5 g/L NHNO, and 80 mM/60 mM NaHPO/KHPO, the strain WX02-Psrflch produced 2,149 mg/L lichenysin, a 16.8-fold improvement compared to that of wild strain WX-02. [ABSTRACT FROM AUTHOR]

Keeping an appropriate mixing state of the multiphase flows in photobioreactors (PBRs) is a key issue for the optimal design and operation of the PBRs. In the present study, an experimental investigation is conducted to quantify the turbulent mixing of multiphase flows inside a flat-panel PBR and its consequential effects on the performance of the PBR for algae cultivation. While a high-resolution particle image velocity (PIV) system is used to achieve detailed flow field measurements to quantify the unsteady behaviors of the multiphase flows and turbulent mixing inside the PBR, algae cultures are also grown in the same PBR under the same test conditions. Detailed flow field measurement results are correlated with the algae growth performance in order to elucidate the underlying physics and explore/optimize design paradigms. The measurement results reveal that even though the airflow rate that is supplied to the PBR plays a dominant role in determining the characteristics of the turbulent mixing in the PBR, the geometric positioning of the aeration inlets also significantly contributes to the turbulent mixing. These differences in turbulent mixing cause differences in algae productivity within the PBR, clearly effecting efficiency of the PBR. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6, n-3), eicosapentaenoic acid (EPA, 20:5, n-3), and arachidonic acid (ARA, 20:4 n-6), have multiple beneficial effects on human health and can be used as an important ingredient in dietary supplements, food, feed and pharmaceuticals. A variety of microorganisms has been used for commercial production of these fatty acids. The microorganisms in the Pythium family, particularly Pythium irregulare, are potential EPA producers. The aim of this work is to provide a safety assessment of P. irregulare so that the EPA derived from this species can be potentially used in various commercial applications. The genus Pythium has been widely recognized as a plant pathogen by infecting roots and colonizing the vascular tissues of various plants such as soybeans, corn and various vegetables. However, the majority of the Pythium species (including P. irregulare) have not been reported to infect mammals including humans. The only species among the Pythium family that infects mammals is P. insidiosum. There also have been no reports showing P. irregulare to contain mycotoxins or cause potentially allergenic responses in humans. Based on the safety assessment, we conclude that P. irregulare can be considered a safe source of biomass and EPA-containing oil for use as ingredients in dietary supplements, food, feed and pharmaceuticals. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis. [ABSTRACT FROM AUTHOR]