Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeş-Bolyai University, Cluj-Napoca, Romania.
Molecular Biology Center, Institute for Interdisciplinary Research in Bio-Nano-Sciences, Babeş-Bolyai University, Cluj-Napoca, Romania.
J Appl Microbiol. 2018 Nov;125(5):1343-1357. doi: 10.1111/jam.14029. Epub 2018 Aug 14.
This study aimed at unprecedented physical and chemical evaluation of the 'green plastics' polyhydroxyalkanoates (PHAs), in an extremely halotolerant Halomonas elongata strain 2FF under high-salt concentration.
The investigated bacterial strain was isolated from the surface water of the hypersaline Fără Fund Lake. The 16S rRNA gene sequence phylogeny and phenotypic analysis indicated that the isolate belonged to H. elongata. PHA inclusions were observed by Sudan Black B, Nile Red staining, and transmission electron microscopy during growth at high salinity (10%, w/v, NaCl) on 1% (w/v) d-glucose. The produced polymer was quantitatively and qualitatively assessed using crotonic acid assay, elemental analysis, Fourier transform infrared and Raman spectroscopies. Additionally, X-ray powder diffraction, H-NMR spectroscopy, and differential scanning calorimetry were applied. The investigations showed that the intracellular polymer was polyhydroxybutyrate (PHB) of which the strain produced up to 40 wt% of total cell dry weight after 48 h. The analysis of phaC gene from the isolated H. elongata strain indicated that the encoded PHA synthase belongs to Class I PHA synthase family.
Overall, our investigations pointed out that the halotolerant H. elongata strain 2FF was capable to produce significant amounts of PHB from d-glucose, and PHAs from various carbon substrates at high-salt concentrations.
The tested strain showed the ability for significant production of natural, biodegradable polymers under nutrient limitation and hypersaline conditions suggesting its potentiality for further metabolic and molecular investigations towards enhanced biopolymer production. Additionally, this study reports on the unprecedented use of Raman and XPRD techniques to investigate PHAs of an extremely halotolerant bacterium, thus expanding the repertoire of physical methods to study green plastics derived from extremophilic microorganisms.
本研究旨在对极端耐盐的盐单胞菌（Halomonas elongata）2FF 菌株在高盐浓度下前所未有的物理和化学评估“绿色塑料”聚羟基烷酸酯（PHA）。
从高盐度（Fără Fund 湖表面水）中分离出研究用的细菌菌株。16S rRNA 基因序列系统发育和表型分析表明，该分离株属于 elongata 盐单胞菌。在 1%（w/v）d-葡萄糖上高盐（10%，w/v，NaCl）生长时，用苏丹黑 B、尼罗红染色和透射电子显微镜观察到 PHA 内含物。使用巴豆酸测定法、元素分析、傅里叶变换红外和拉曼光谱法对产生的聚合物进行定量和定性评估。此外，还应用了 X 射线粉末衍射、1H-NMR 光谱和差示扫描量热法。研究表明，细胞内聚合物为聚羟基丁酸酯（PHB），该菌株在 48 小时后产生高达 40wt%的总细胞干重。从分离出的 elongata 盐单胞菌phaC 基因分析表明，编码 PHA 合酶属于 I 类 PHA 合酶家族。
总的来说，我们的研究表明，耐盐 elongata 盐单胞菌 2FF 能够在高盐浓度下从 d-葡萄糖和各种碳源中生产大量 PHB 和 PHA。
测试菌株显示出在营养限制和高盐条件下大量生产天然可生物降解聚合物的能力，这表明其具有进一步进行代谢和分子研究以提高生物聚合物产量的潜力。此外，本研究报告了首次使用拉曼和 XPRD 技术研究极端耐盐细菌的 PHAs，从而扩展了用于研究源自极端微生物的绿色塑料的物理方法 repertoire。
