Recent advancements in bioreactions of cellular and cell-free systems: A study of bacterial cellulose as a model

Conventional approaches of regulating natural biochemical and biological processes are greatly hampered by the complexity of natural systems. Therefore, current biotechnological research is focused on improving biological systems and processes using advanced technologies such as genetic and metabolic engineering. These technologies, which employ principles of synthetic and systems biology, are greatly motivated by the diversity of living organisms to improve biological processes and allow the manipulation and reprogramming of target bioreactions and cellular systems. This review describes recent developments in cell biology, as well as genetic and metabolic engineering, and their role in enhancing biological processes. In particular, we illustrate recent advancements in genetic and metabolic engineering with respect to the production of bacterial cellulose (BC) using the model systems Gluconacetobacter xylinum and Gluconacetobacter hansenii. Besides, the cell-free enzyme system, representing the latest engineering strategies, has been comprehensively described. The content covered in the current review will lead readers to get an insight into developing novel metabolic pathways and engineering novel strains for enhanced production of BC and other bioproducts formation.     

Systematic Understanding of Recent Developments in Bacterial Cellulose Biosynthesis at Genetic,Bioprocess and Product Levels

Engineering biological processes has become a standard approach to produce various commercially valuable chemicals, therapeutics, and biomaterials. Among these products, bacterial cellulose represents major advances to biomedical and healthcare applications. In comparison to properties of plant cellulose, bacterial cellulose (BC) shows distinctive characteristics such as a high purity, high water retention, and biocompatibility. However, low product yield and extensive cultivation times have been the main challenges in the large-scale production of BC. For decades, studies focused on optimization of cellulose production through modification of culturing strategies and conditions. With an increasing demand for BC, researchers are now exploring to improve BC production and functionality at different categories: genetic, bioprocess, and product levels as well as model driven approaches targeting each of these categories. This comprehensive review discusses the progress in BC platforms categorizing the most recent advancements under different research focuses and provides systematic understanding of the progress in BC biosynthesis. The aim of this review is to present the potential of ‘modern genetic engineering tools’ and ‘model-driven approaches’ on improving the yield of BC, altering the properties, and adding new functionality. We also provide insights for the future perspectives and potential approaches to promote BC use in biomedical applications.     

Physicochemical properties of bacterial cellulose obtained from different Kombucha fermentation conditions

The production of bacterial cellulose has been limited due to its high cost and low productivity. Alternative low-cost sources of this biopolymer of high purity and biocompatibility are needed in order to benefit from its enormous potential. Kombucha tea is a trend functional beverage whose production is growing exponentially worldwide, and the bacteria present in this fermented beverage belonging to the genus Komagataeibacter are capable of producing a crystalline biofilm with interesting properties. Obtaining bacterial cellulose from Kombucha tea has already been studied, however several fermentation conditions are being optimized in order to scale-up its production. In this study, we characterized the bacterial cellulose produced from three different Kombucha fermentation conditions. The scanning electron microscopy images revealed the crystalline structure of the biofilms. The energy-dispersive x-ray analysis exhibited the chemical composition of the crystals. The thermogravimetric analysis showed a rate of degradation between 490 and 560°C and the differential scanning calorimetry confirmed the presence of crystalline and amorphous regions in the bacterial cellulose samples. The results suggested that crystalline cellulose could be obtained by varying the fermentation conditions of Kombucha tea.     

细菌纤维素的生产研究进展

介绍了细菌纤维素(BC)的特殊结构、功能、物理和化学性质以及应用前景。综述了目前细菌纤维素的生产菌种、生产方式及原料的研究进展。     

细菌纤维素合成与鉴定研究综述

细菌纤维素（BC）因其独特性能被广泛应用于医药、食品等领域，目前其高产量菌株筛选、合成成本降低及合成途径改良等成为研究热点。本文依据国内外文献并结合团队研究成果对BC合成与鉴定的相关研究进行综述。首先对BC合成菌筛选及碳源利用的研究进行了分析，总结了降低BC合成成本的研究思路。其次对鉴定菌株合成产物的方法进行了归纳，总结了不同方法的特点。然后结合本团队筛选出的BC生产菌XJL-06-4 BC合成酶基因分析结果，综述了BC合成途径、合成酶存在形式以及基因水平调控作用，为BC在分子水平上通过改变合成途径提高产量提供新思路。最后，总结BC微生物发酵生产存在的问题，多角度提出解决方案。     

细菌纤维素纳米纤维的细胞相容性评价

细菌纤维素是一种天然的纳米纤维材料,在组织工程材料领域具有广阔的应用前景。在前期研究的基础上,以细菌纤维素(BC)及细菌纤维素/聚丙烯酰胺双网络水凝胶(BC/PAM)复合材料为研究对象,大鼠成纤维细胞L929及血管内皮细胞为细胞模型,采用扫描电子显微镜观察细胞在材料上的黏附形态,并通过MTT法对细胞的增殖行为进行评价,以此考察BC及BC/PAM复合材料的细胞相容性,初步评价上述纳米纤维作为组织工程材料的应用可能性。结果表明,内皮细胞在纯BC材料上表现出良好的黏附形态和增殖行为,而成纤维细胞在纯BC及BC/PAM复合材料上的增殖趋势均低于空白对照组。     

达托霉素及其衍生物生物合成的研究进展

随着高致病耐药菌的不断出现,临床上对新抗生素的需求十分迫切。达托霉素作为环脂肽类抗生素的第一个产品对耐药菌有很好的杀菌效果,且制剂用药方便,毒副作用小,被公认为病原菌最后一道防线--万古霉素的最佳替代品。本文在介绍达托霉素的理化特征和抗菌活性、合成基因簇的研究成果的基础上,重点对达托霉素及其衍生物的研究状况进行了综述,对达托霉素的生产及其衍生物的开发进行了展望：结合系统生物学为已有的达托霉素产生菌进一步进行代谢工程改造提供指导;通过合成生物学使达托霉素衍生物成为新型抗生素。     

Some properties of bacterial cellulose produced by new native strain Gluconacetobacter sp. A06O2 obtained from Turkish vinegar

The aim of the study was to isolate and identify an acetic acid bacterial strain having high cellulose yield and to investigate some physicochemical properties of bacterial cellulose (BC). Acetic acid bacteria were isolated by using 62 samples (vinegar, fruit, vegetable, and soil) from different region of Turkey. The cellulose production ability of 153 isolates was determined. A strain (A06O2) having high and stable cellulose yield was identified by biochemical tests and 16S rRNA gene sequencing and compared with type strain Gluconacetobacter xylinus NRRL B‐759. Based on the results, strain A06O2 was named at the genus level as Gluconacetobacter, however, species level identification could not be made. Celluloses from both strains were purified to investigate the physicochemical properties such as thermal properties, solubility in various solvents, elemental composition, tensile properties, and surface properties by FTIR and SEM. The results showed that the cellulose samples of two bacterial strains differed in the physicochemical properties. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

小径纳米纤维素管的生物制备及其表征

以透氧硅胶双套管为模具,设计组装了一套新型生物反应装置,利用红茶菌和套管法生产细菌纤维素（BC）小径人工血管材料。研究发现,细菌纤维素可以分别在内外两根硅胶管的外表面和内表面同时生成,并最终长到一起形成整体BC管。获得的BC管不仅具有较好的强度和光滑平整的内外表面,而且纳米纤维交织紧密,无纤维分层现象,具有突出优点和应用潜力。该制备方法简便易行,成本低廉,生产效率高,可工业化生产。     

剪切力对木葡糖醋杆菌及细菌纤维素合成的影响

以摇瓶和发酵罐两种培养体系为对象,考察了剪切力对木葡糖醋杆菌生长和细菌纤维素合成的影响。结果表明,剪切力的存在对细菌纤维素的合成不利,在添加玻璃珠的三角瓶中经9轮震荡培养后,细菌纤维素的产量降至原始菌株的23．6％;在机械搅拌罐中培养时,用剪切力大的六叶平桨进行发酵,细菌纤维素的产量最低,而用转速降低的框式桨进行发酵,细菌纤维素的产量较高。剪切力也影响木葡糖醋杆菌的形态和生长周期,剪切力的存在使细菌菌体变小,单位体积发酵液菌浓降低,菌落形态改变,菌株进入对数生长期的时间延后。实验结果为今后改进提高细菌纤维素动态培养产量提供了理论依据。     

The Simple Method of Preparation of Highly Carboxylated Bacterial Cellulose with Ni- and Mg-Ferrite-Based Versatile Magnetic Carrier for Enzyme Immobilization

The bacterial cellulose (BC) is a versatile biopolymer of microbial origin characterized by high purity and unusual water and material properties. However, the native BC contains a low number of functional groups, which significantly limits its further application. The main goal of its effective modification is to use methods that allow the unusual properties of BC to be retained and the desired functional group to be efficiently introduced. In the present study, the new magnetic carrier based on functionalized citric acid (CA) bacterial cellulose was developed and tested to support critical industrial enzymes such as lipase B from Candida antarctica and phospholipase A from Aspergillus oryzae. The applied method allowed BC to be effectively modified by citric acid and a sufficient number of carboxylic groups to be introduced, up to 3.6 mmol of COOH per gram of dry mass of the prepared carrier. The DSC and TGA analyses revealed carrier stability at operational temperatures in the range of 20 °C to 100 °C and substantially influenced the amount of the introduced carboxyl groups on carrier properties. Both enzymes’ immobilization significantly improves their thermal stability at 60 °C without a significant thermal and pH optima effect. The analyzed enzymes showed good operational stability with a significant residual activity after ten cycles of repeated uses. The new magnetic carrier based on highly carboxylated bacterial cellulose has a high application capability as matrix for immobilization the various enzymes of industrial interest.     

细菌纤维素发酵原料的研究进展

细菌纤维素是一种新型微生物合成材料,在食品、造纸、纺织、生物医药、声学器材振动膜和功能复合材料等方面均有很好的应用前景。细菌纤维素发酵培养基(尤其碳源)的成本是现今制约细菌纤维素推广应用的主要因素之一。甘露醇、果糖和葡萄糖等合成培养基所用碳源因其价格较高仅适用于实验室研究和小型发酵生产,规模化生产细菌纤维素的潜在原料应是一些量大价低的天然原料,包括水果类原料、糖质原料、低值淀粉类原料和废弃纤维素类原料等。木质纤维素原料是最具发展潜力的细菌纤维素碳源,也是细菌纤维素产业的根本出路,但目前存在一些技术瓶颈,制约了其开发利用,是一远期战略目标。文章简要介绍了细菌纤维素的基本情况,系统阐述了国内外发酵生产细菌纤维素原料的研究进展,展望了今后的发展趋势。     

发酵法生产1,3-丙二醇的高产策略

以1,3-丙二醇为单体生产的聚对苯二甲酸二醇酯是一种新型聚酯材料,具有优良的特性,是目前合成纤维新品种开发热点。1,3-丙二醇工业生产主要采用化学法合成,设备投资大、技术难度高、需要重金属催化剂、污染环境,各国都在致力于开发生物发酵法生产技术,由DuPont,Tate&Lyle,Genencor联合建立的年产10万磅的中试发酵装置已投入运转。本文详细介绍了甘油在微生物体内的代谢途径、不同菌种的代谢特点、基因工程研究进展,指出发酵法生产1,3-丙二醇的高产策略是筛选菌种、优化培养工艺、混合菌发酵、双底物发酵和构建基因工程菌。     

Bacterial Cellulose Membranes Used as Artificial Substitutes for Dural Defection in Rabbits

To improve the efficacy and safety of dural repair in neurosurgical procedures, a new dural material derived from bacterial cellulose (BC) was evaluated in a rabbit model with dural defects. We prepared artificial dura mater using bacterial cellulose which was incubated and fermented from Acetobacter xylinum. The dural defects of the rabbit model were repaired with BC membranes. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were humanely euthanized by intravenous injection of phenobarbitone, at each time point, after the operation. Then, the histocompatibility and inflammatory effects of BC were examined by histological examination, real-time fluorescent quantitative polymerase chain reaction (PCR) and Western Blot. BC membranes evenly covered the surface of brain without adhesion. There were seldom inflammatory cells surrounding the membrane during the early postoperative period. The expression of inflammatory cytokines IL-1β, IL-6 and TNF-α as well as iNOS and COX-2 were lower in the BC group compared to the control group at 7, 14 and 21 days after implantation. BC can repair dural defects in rabbit and has a decreased inflammatory response compared to traditional materials. However, the long-term effects need to be validated in larger animals.     

Synthetic Phosphoethanolamine Cellobiose Promotes Escherichia coli Biofilm Formation and Congo Red Binding

Urinary tract infections (UTIs) are caused by bacteria growing in complex, multicellular enclosed aggregates known as biofilms. Recently, a zwitterionic cellulose derivative produced in Escherichia coli (E. coli) was determined to play an important role in the formation and assembly of biofilms. In order to produce a minimal, yet structurally defined tool compound to probe the biology of the naturally occurring polymer, we have synthesized a zwitterionic phosphoethanolamine cellobiose (pEtN cellobiose) and evaluated its biofilm activity in the Gram-negative bacterium E. coli, a pathogen implicated in the pathogenesis of UTIs. The impact of synthetic pEtN cellobiose on biofilm formation was examined via colorimetric assays which revealed an increase in cellular adhesion to an abiotic substrate compared to untreated samples. Additionally, Congo red binding assays indicate that culturing E. coli in the presence of pEtN cellobiose enhances Congo Red binding to bacterial cells. These results reveal new opportunities to study the impact glycopolymers have on cellular adhesion in Gram-negative pathogens.     

Small Molecule Inhibitors of AI-2 Signaling in Bacteria: State-of-the-Art and Future Perspectives for Anti-Quorum Sensing Agents

Bacteria respond to different small molecules that are produced by other neighboring bacteria. These molecules, called autoinducers, are classified as intraspecies (i.e., molecules produced and perceived by the same bacterial species) or interspecies (molecules that are produced and sensed between different bacterial species). AI-2 has been proposed as an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production and biofilm formation in some bacteria, including bacteria of clinical relevance. Several groups have embarked on the development of small molecules that could be used to perturb AI-2 signaling in bacteria, with the ultimate goal that these molecules could be used to inhibit bacterial virulence and biofilm formation. Additionally, these molecules have the potential to be used in synthetic biology applications whereby these small molecules are used as inputs to switch on and off AI-2 receptors. In this review, we highlight the state-of-the-art in the development of small molecules that perturb AI-2 signaling in bacteria and offer our perspective on the future development and applications of these classes of molecules.     

一体化生物加工过程生产乙醇的研究进展

一体化生物加工过程（consolidated bioprocessing,CBP）指通过对理想底盘微生物的开发和利用来实现一步转化木质纤维素为生物产品的生物加工程序。本文回顾了一体化生物加工过程的研究背景,简述了其开发理念和技术路线,全面综述了近年来该技术在转化木质纤维素生产二代生物乙醇研究中的不同策略及最新的研究进展。分析了CBP系统中自然菌株、重组菌株和共培养菌株在转化木质纤维素生产生物乙醇时的优点和瓶颈因素。研究了基因工程、代谢工程等工程手段和技术在克服此技术中的阻碍性因素及提升乙醇得率等方面的应用价值和潜力。最后,论述了组学及合成生物学等新兴生物技术对CBP生物乙醇的贡献和二代生物乙醇的商业化发展现状及CBP乙醇未来所面临的机遇与挑战。     

Improvement of bacterial cellulose production in <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> using byproduct produced by <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis>

A single sugar α-linked glucuronic acid based oligosaccharide (SSGO) is water soluble oligosaccharides (WSOS) obtained by Gluconacetobacter hansenii PJK (KCTC 10505BP) as a byproduct during bacterial cellulose (BC) production. In this study, SSGO was used for the improvement of BC production by the vinegar bacterium, Acetobacter xylinum, which produces heteropolysaccharides as a byproduct. The addition of 1.0% SSGO to the chemically defined medium (CDM) resulted in an 89.3% increase in BC production by A. xylinum after 15 days of cultivation under static condition, and a 52.3% increase in BC production by G. hansenii. Both the dry cell weight and live cell density of A. xylinum increased 50% with the addition of 1.0% SSGO. SSGO successfully improved BC production by A. xylinum.     

Potato Juice,a Starch Industry Waste,as a Cost-Effective Medium for the Biosynthesis of Bacterial Cellulose

In this work, we verified the possibility of valorizing a major waste product of the potato starch industry, potato tuber juice (PJ). We obtained a cost-effective, ecological-friendly microbiological medium that yielded bacterial cellulose (BC) with properties equivalent to those from conventional commercial Hestrin–Schramm medium. The BC yield from the PJ medium (>4 g/L) was comparable, despite the lack of any pre-treatment. Likewise, the macro- and microstructure, physicochemical parameters, and chemical composition showed no significant differences between PJ and control BC. Importantly, the BC obtained from PJ was not cytotoxic against fibroblast cell line L929 in vitro and did not contain any hard-to-remove impurities. The PJ-BC soaked with antiseptic exerted a similar antimicrobial effect against Staphylococcus aureus and Pseudomonas aeruginosa as to BC obtained in the conventional medium and supplemented with antiseptic. These are very important aspects from an application standpoint, particularly in biomedicine. Therefore, we conclude that using PJ for BC biosynthesis is a path toward significant valorization of an environmentally problematic waste product of the starch industry, but also toward a significant drop in BC production costs, enabling wider application of this biopolymer in biomedicine.     

甲基营养型大肠杆菌构建策略的研究进展

利用微生物细胞工厂实现原料转化和产品合成是绿色生物制造的核心。然而,当前生物制造仍以富含糖类的谷物粮食为主要原料,存在“与民争粮”的争议,亟需开发非粮原料。甲醇作为煤化工产业中的重要产品,具有来源广、价格低、还原性强等优势,有替代粮食原料的潜力。天然甲基营养菌可利用甲醇生产单细胞蛋白和各种氨基酸,但存在理论收率低、遗传改造工具不足等缺点。随着合成生物学的发展,以大肠杆菌等模式生物作为底盘细胞构建人工甲基营养菌,实现甲醇到各种化学品的生产已成为研究热点。本文总结了多种甲基营养型大肠杆菌的构建策略,明确了影响天然路径代谢的关键因素与代谢过程中的重要中间产物,概括了各种天然路径在大肠杆菌中的优化策略与人工新路径的构建方法,并对工程菌株的优化提出了展望。