Morphological Study of PHA Producing Bacteria

Microbial cells produce a variety of storage materials, which serve as an energy source, carbon source, etc. Polyhydroxyalkanoates (PHA), polyesters of hydroxyalkanoic acids, are biodegradable plastics accumulated as granules in cells of many prokaryotic microorganisms. Primarily PHA are used in microbial cells as storage material, however, recent studies revealed, that cells containing PHA are also more resistant to stress environment such as osmotic imbalances, UV irradiance or temperature changes. Production of polyhydroxyalkanoates is induced in excess of carbon source and also limitation of another element essential for cellular growth. Besides microbial cell storage material, PHA are also considered as a promising bioplastic material, due to its biodegradability and mechanical properties similar to petrochemical plastics, such as polypropylene (PP) [1–3]. Currently, Cupriavidus necator is considered as one of the biggest PHA producers. Unfortunately, the production of PHA using heterotrophic microorganisms requires large amounts of carbon substrates, such as glucose or fructose, which makes the production quite expensive compared to PP. Carbon substrates could make up to 50% of all production costs. One of the possibilities, how to reduce these expenses, could be the use of waste material (eg. frying oils) as a carbon source or production of PHA by photoautotrophic microorganisms [1, 3]. Bacterial cells were fixed using high-pressure freezing method. After fixation samples were processed using either freeze-fracture method for observation in cryo-SEM or freeze substitution followed by embedding in epoxy-resin for observation in TEM. Freeze fracturing method enables the imaging of intracellular content of cells. PHA granules at temperatures of −130 °C remain elastic and can be observed in cryo-SEM sticking out of fractured cells. Serial block-face scanning electron microscopy (SBFSEM) is a method to generate high resolution three-dimensional images from small samples. SBFSEM microscope consists of an ultramicrotome mounted inside the vacuum chamber of a scanning electron microscope. Samples are prepared by methods similar to that in TEM. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. After SFBSEM imaging we improved the image quality for a segmentation of cells and PHA granules using filtration via anisotropic diffusion in ImageJ. The contrast and brightness were adjusted to emphasize the differences between image background and regions of interest. Image segmentation was performed using the AMIRA software. Our comprehensive morphological study indicates that the method involving cryo-SEM imaging in combination with high-pressure HPF freezing and perpendicular freeze-fracturing, is an excellent technique for analysing highly hydrated samples. Cells containing polymer particles, which are elastic even at very low temperatures, were visualized using this method. By TEM it is able to monitor the distribution of PHB granules inside the microbial cells. SBFSEM provides spatial information. A comparison of the morphology of the internal and external cell structure shows that the Cupriavidus necator H16 contains significant amounts of PHA granules, which are distributed throughout the whole cell volume [4]. C. necator containing PHB particles A) cryo-SEM micrographs shows freeze-fracture face; B) TEM micrographs, white areas are PHB particles; C) 3D reconstruction (SBFSEM, Amira soft.).