CRISPR/dCas9干扰bcsD基因表达调控细菌纤维素结构

木葡糖酸醋杆菌(Gluconacetobacter xylinus)是细菌纤维素的主要生产菌株。在该菌中,BcsD是纤维素合酶的亚基之一,参与细菌纤维素的组装过程。利用CRISPR/dCas9系统调控bcsD基因的表达量,获得了一系列bcsD基因表达量不同的木葡糖酸醋杆菌。通过分析细菌纤维素的结构特征发现,细菌纤维素的结晶度和孔隙率随着木葡糖酸醋杆菌中bcsD表达量的变化而发生改变。其中孔隙率的变化范围在59.95%–84.05%之间,结晶度的变化范围在74.26%–93.75%之间,而细菌纤维素的产量并未因bcsD的表达量变化而发生显著下降。结果表明,bcsD的表达量低于55.34%后,细菌纤维素的孔隙率显著上升,并且细菌纤维素的结晶度与bcsD的表达量呈正相关。最终,通过干扰bcsD基因的表达,实现了一步发酵木葡糖酸醋杆菌获得了产量稳定且结构不同的细菌纤维素。     

细菌纤维素在医学方面的应用

细菌纤维素是由木葡糖酸醋杆菌等细菌合成的纤维素,在化学组成、分子结构上与植物纤维素相近,但具有传统的纤维素所无法比拟的优势,如高亲水性、持水性、生物适应性、可调控性以及高纯度、高透明度等,因而在医学上显示出了巨大的应用潜力。细菌纤维素可用作人造皮肤、外科敷料、人造血管、软骨组织、震动膜、缓释载体等,是最有前途的生物聚合材料之一。     

表达淀粉酶、糖化酶葡糖酸醋杆菌工程菌的构建

采用基因融合技术,将葡糖酸醋杆菌Gluconacetobacter hansenii ATCC23769分泌蛋白CMCax的信号肽序列分别与来源于枯草芽胞杆菌的淀粉酶基因、黑曲霉的糖化酶基因融合构建融合蛋白,连入能在G.hansenii ATCC23769自主复制的载体pbs-H1S中,电击转入G.hansenii ATCC23769,构建能内源表达淀粉酶、糖化酶,以及淀粉酶-糖化酶的葡糖酸醋杆菌。淀粉平板透明圈检测结果和DNS测酶活结果显示,构建的3种工程菌能成功表达并分泌淀粉酶和糖化酶。     

一株产细菌纤维素菌株的筛选鉴定及其产物分析

从红茶菌液中筛选获得一株产细菌纤维素的菌株BC-41,经生理生化分析和分子生物学鉴定,现证实该菌株为中间葡糖酸醋杆菌(Gluconacetobacter intermedius)。对该菌株所产生的细菌纤维素进行了物理特性的表征和分析,获得以下数据:BC-41所产的纤维素纯度达到91.32%,湿纤维素膜含水率达99.16%,每克干纤维素膜能吸水28.59 g;扫描电子显微镜观察,显示该纤维素具有网状结构,且纤维束宽度分布在40-100 nm之间;X射线衍射分析,证实该纤维素的晶型为纤维素I型,结晶指数为48.8%;通过黏度测定法,得出该纤维素的平均聚合度达2 100。     

福建红曲醋中产酸菌株的分离及鉴定

添加含有高浓度乙醇的红曲酒至福建传统红曲醋醋母中富集产酸菌株,采用高浓度乙醇平板,依据溶解圈指标从福建传统红曲醋液体循环工艺样品中分离出7株产酸菌株。综合菌株形态、生理生化实验以及16S r DNA序列测定等信息,确定这7株菌株分类地位为变形菌门(Proteobacteria)α-变形菌纲(Alphaproteobacteria)红螺菌目(Rhodospirillales)醋酸菌科(Acetobacteraceae)葡糖酸醋杆菌属(Gluconacetobacter),其中菌株Y5052、Y5054、Y5072、Y5092鉴定为斯氏葡糖酸醋杆菌(Gluconacetobacter swingsii);菌株Y5032、Y5033、Y5071鉴定为欧洲葡糖酸醋杆菌(Gluconacetobacter europaeus)。测定这些菌株的产酸能力,菌株Y5052产酸量最低,7 d达15 g/L;菌株Y5054产酸能力最强,7 d产酸量达57.0 g/L。     

细菌纤维素的研究进展

细菌纤维素是由醋酸杆菌属、根瘤菌属、土壤杆菌属、八叠球菌属等的某些细菌在一定条件下产生的,其中最有代表性的细菌是木醋杆菌。与传统植物纤维素相比,细菌纤维素具有很高的化学纯度。主要介绍细菌纤维素性质、生物合成的方法及其在食品工业、造纸工业和作为一种生物材料在医学工程等方面的应用。     

产细菌纤维素Axy-Ⅰ菌株的鉴定及产物分析

目的：对1株产细菌纤维素的菌株Axy-I进行鉴定,并对其在不同培养条件下的产物进行分析。方法：通过生理生化检测和16S rDNA序列分析,对菌株Axy-I进行鉴定;比较静态培养和摇瓶动态培养7d后所产细菌纤维素的产率,并利用扫描电镜观察产物的超微观结构特征。结果：菌株Axy-I的形态、菌落特征和生理生化特性与葡糖酸醋酸杆菌属一致,16S rRNA序列长度为1436bp,与Gluconacetobacter sp.4L（Genbank登录号为：AY741144.1）的同源性达97%。静态培养细菌纤维素得率为4.72g/L,纤维直径82%分布在30-80nm之间,动态培养得率为8.12g/L,纤维直径90%分布在30～70nm之间。结论：菌株Axy-I属于葡糖酸醋酸杆菌属,动态培养所产的细菌纤维素纤维丝比静态更纤细,产率也更高。     

细菌纤维素生产菌株的分离和菌种初步鉴定

从长膜的醋醅中分离出一株发酵生产细菌纤维素产量较高且稳定的醋酸菌M12。根据《常见细菌系统鉴定手册》和《伯杰氏细菌鉴定手册》第九版,对醋酸菌M12进行了形态和生理生化特征的分析、测定了G＋Cmol％含量,初步鉴定该菌为醋化醋杆菌木质亚种（Acetobacter xylinum subsp.xylinum,又称木醋杆菌）。     

木醋杆菌纤维素合成操纵子的克隆及棉花转化

革兰氏阴性菌木醋杆菌 (Acetobacterxylinum (Brown)Yamada)合成一种由纤维素微纤丝组成的胞外带状物。与高等植物纤维素相比 ,它具有独特的结构和机械性能。根据从木醋杆菌ATCC 5 35 82克隆的acs纤维素合成操纵子序列设计引物 ,用PCR的方法从木醋杆菌Ay2 0 1中克隆了ayacs纤维素合成操纵子的全部 4个基因。序列比较发现 ,两者高度同源。将连上CaMV 35S启动子的acsA、acsB克隆到植物表达载体pCAMBIA 130 1上 ,acsC、acsD克隆到pCOB30 2_3中。然后通过花粉管通道法转化棉花 (Gossypiumhirsutum)胚珠 ,收获的种子在含有卡那霉素和除草剂的双抗培养基上进行筛选。PCR检测发现 934粒种子中有 5棵植株含有全部 4个基因。这是首次将编码 4个功能蛋白的细菌操纵子成功地转入棉花     

利用西瓜汁合成细菌纤维素的研究

利用木醋杆菌为实验菌种,通过选择不同的种龄、接种量、发酵培养基液面积/液体积、初始pH值、培养温度和时间,对西瓜汁合成细菌纤维素的发酵务件进行研究,得到了最佳培养条件,并利用扫描电镜、红外光谱、元素分析等手段对合成细菌纤维素的微结构进行了观察分析.     

Increased yield and selected properties of bacterial cellulose exposed to different modes of a rotating magnetic field

Rotating magnetic field (RMF) is an interesting alternative to conventional bacterial cellulose (BC) production methods. The BC synthesis processes may be affected by RMF, which facilitates the transfer of oxygen and nutrients from the media to the microbial cells. RMF may also directly influence the various physical and chemical properties of BC. The main aim of the present study was to evaluate the impact of the RMF on the BC in regard to its yield and material properties. The correlation between the efficiency of polymer production and the different time of exposure to the RMF was also analyzed to determine the conditions of lower energy consumption during the cellulose formation process. It was found that the Gluconacetobacter xylinus cultures exposed to the RMF for a half of the time of the entire cellulose production process (72 h), considering the results obtained in controls, synthesized BC more effectively than bacteria continuously exposed to the RMF for 144 h. Furthermore, the application of the RMF, regardless of the exposure mode, did not negatively affect the polymer material properties. It was concluded that the use of the RMF may provide a novel technique for altering cellulose biogenesis and may be used in multiple biotechnological applications.     

Production of nanocellulose in miniature-bioreactor: Optimization and characterization

Bacterial cellulose (BC) is a very fascinating microbial biopolymer which is mainly produced by Gluconacetobacter xylinum. Optimization of BC production by G. xylinum was performed based on scale-down studies in miniature-bioreactor and response surface methodology in which the optimum pH value (6.5) and shaking rate (50?rpm) were obtained. The static culture condition for BC production has newly been defined. Nanostructure of BC includes nanofibers up to (60?nm) and nanoporosity up to (265?nm) was observed by scanning electron microscopy. By Fourier transform infrared spectroscopy study, the most expected BC interaction is nucleophilic interaction. MTT assay showed high biocompatibility. Appropriate mechanical strength (0.37?MPa) and Young’s modulus (3.36?MPa) evinced BC scaffold utilization for skin tissue. The results indicate that BC sheets can be utilized in biomedical application and nanotechnology approaches.     

Recent advances in bacterial cellulose production

Bacterial cellulose (BC), which is produced by some bacteria, has unique structural, functional, physical and chemical properties. Thus, the mass production of BC for industrial application has recently attracted considerable attention. To enhance BC production, two aspects have been considered, namely, the engineering and genetic viewpoints. The former includes the reactor design, nutrient selection, process control and optimization; and the latter the cloning of the BC synthesis gene, and the genetic modification of the speculated genes for higher BC production. In this review, recent advances in BC production from the two viewpoints mentioned above are described, mainly using the bacteriumGluconacetobacter xylinus.     

Bacterial cellulose synthesized with apple pomace enhanced by ionic liquid pretreatment

Abstract

Apple pomace was explored as alternative feedstock for producing bacterial cellulose (BC) by Gluconacetobacter xylinus following a cellulase saccharification performed after pretreatment of 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). The dissolving process of apple pomace cellulose was observed by polarized light microscopy (PLM). As FT-IR and XRD results demonstrated, the IL pretreatment proved to be a physical process and no changes in the crystalline structure occurred during the pretreatment. However, the SEM result showed that more fissures and breakages appeared on the surface of pomace microfibers after IL-pretreating, which increased the contact area with cellulase and improved the enzymatic hydrolysis efficiency. An enhancing effect on the BC yield has been observed, 27% higher yield of BC obtained from hydrolysate as compared to sucrose-based medium indicates efficiency of IL-treated apple pomace to serve as high quality feedstock in BC production.     

Simple fed-batch cultivation strategy for the enhanced production of a single-sugar glucuronic acid-based oligosaccharides by a cellulose-producing <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> strain

The production of water-soluble single-sugar glucuronic acid-based oligosaccharides (WSOS) by a cellulose producing strain Gluconacetobacter hansenii PJK was studied in a periodically recycled and fed-batch cultivations using glucose/ethanol or glucose only. Fermentations were carried out in a 2 L jar fermenter equipped with a turbine impeller with 6 flat blades. WSOS were produced constantly but the bacterial cellulose (BC) production stopped at 48 h of cultivation in a periodically recycled culture using the exhausted medium supplemented with glucose and ethanol. Tremendous quantities of WSOS were obtained in fed-batch cultivations using glucose/ethanol (35.6 g/L at 132 h of cultivation) or glucose only (86 g/L after 240 h of cultivation) as the nutritional source. However, the BC production yield under these nutritional conditions decreased significantly in comparison to previous studies about the BC production by the same strain. The overall results revealed that G. hansenii is capable of producing enormous quantities of WSOS compared to those reported previously for compounds of a related chemical nature. Moreover, the WSOS production was found to be dependent on the pH of the culture broth.     

Efficient bioconversion from acid hydrolysate of waste oleaginous yeast biomass after microbial oil extraction to bacterial cellulose by Komagataeibacter xylinus

Biomass acid hydrolysate of oleaginous yeast Trichosporon cutaneum after microbial oil extraction was applied as substrate for bacterial cellulose (BC) production by Komagataeibacter xylinus (also named as Gluconacetobacter xylinus previously) for the first time. BC was synthesized in static culture for 10 days, and the maximum BC yield (2.9?g/L) was got at the 4th day of fermentation. Most carbon sources in the substrate (glucose, mannose, formic acid, acetic acid) can be utilized by K. xylinus. The highest chemical oxygen demand (COD) removal (40.7?±?3.0%) was obtained at the 6th day of fermentation, and then the COD increased possibly due to the degradation of BC. The highest BC yield on COD consumption was 38.7?±?4.0% (w/w), suggesting that this is one efficient bioconversion for BC production. The BC structure was affected little by the substrate by comparison with that generated in classical HS medium using field-emission scanning electron microscope (FE-SEM), Fourier transform infrared, and X-ray diffraction. Overall, this technology can both solve the issue of waste oleaginous yeast biomass and produce valuable biopolymer (BC).     

Efficient Using Durian Shell Hydrolysate as Low-Cost Substrate for Bacterial Cellulose Production by <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Durian is one important tropical fruit with high nutritional value, but its shell is usually useless and considered as waste. To explore the efficient and high-value utilization of this agricultural and food waste, in this study, durian shell was simply hydrolyzed by dilute sulfuric acid, and the durian shell hydrolysate after detoxification was used for bacterial cellulose (BC) production by Gluconacetobacter xylinus for the first time. BC was synthesized in static culture for 10 days and the highest BC yield (2.67 g/L) was obtained at the 8th day. The typical carbon sources in the substrate including glucose, xylose, formic acid, acetic acid, etc. can be utilized by G. xylinus. The highest chemical oxygen demand (COD) removal (16.40%) was obtained at the 8th day. The highest BC yield on COD consumption and the highest BC yield on sugar consumption were 93.51% and 22.98% (w/w), respectively, suggesting this is one efficient bioconversion for BC production. Durian shell hydrolysate showed small influence on the BC structure by comparison with the structure of BC generated in traditional Hestrin–Schramm medium detected by FE-SEM, FTIR, and XRD. Overall, this technology can both solve the issue of waste durian shell and produce valuable bio-polymer (BC).     

Metabolic flux analysis of Gluconacetobacter xylinus for bacterial cellulose production

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics.     

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

Production of bacterial cellulose by<Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> PJK isolated from rotten apple

A cellulose-producing strain isolated from rotten apples was identified asGluconace-tobacter hansenii based on its physiological properties and the 16S rDNA complete sequencing method, and specifically namedGluconacetobacter hansenii PJK. The amount of bacterial cellulose (BC) produced byG. hansenii PJK in a shaking incubator was 1.5 times higher than that produced in a static culture. The addition of ethanol to the medium during cultivation enhanced the productivity of bacterial cellulose, plus the supplementation of 1% ethanol into the culture medium made the produced BC aggregate into a big lump and thus protected the bacterial-cellulose-producingG. hansenii PJK cells in the shear stress field from being converted into noncellulose-producing (Cel) mutants. Cells subcultured three times in a medium containing ethanol retained their ability to produce BC without any loss in the production yield.