Synthesis of glycogen by Chlorobium limicola IMV K-8 while growth in wastewater

Due to the high content of organic compounds, the distillery wastewater can be a good substrate for the production of glycogen during cultivation of green photosynthetic bacteria. Green photosynthetic bacteria Chlorobium limicola IMV K-8 are producers of glycogen and show exoelectrogenic properties when grown alone or inside the co-culture with heterotrophic bacteria-exoelectrogens in wastewater of various origins. In our previous works it was found that due to the phototrophic growth of C. limicola IMV K-8 in the distillery wastewater significantly reduces the content of compounds of nitrogen, sulfur, Ca2+, Mg2+ and others. The study of the patterns of glycogen synthesis by green photosynthetic bacteria during growth in such an extreme environment as the wastewater of a distillery has prospects for the development of biotechnology for the production of this polysaccharide. The aim of the study was to investigate the glycogen content in C. limicola IMV K-8 cells under different growth conditions in the wastewater of the distillery. Bacteria were grown in the wastewater of the distillery under light (phototrophic growth) and without light exposure (heterotrophic growth). Bacterial cells grown on GSB medium under light (phototrophic growth) and without light (heterotrophic growth) exposure were used as controls. Glycogen content was determined at 7, 14, 21 and 30 days of growth by the glucose oxidase method. Glucose or glycogen in the wastewater of the distillery without the introduction of bacteria was not detected. It was found that the content of glycogen in cells of C. limicola IMV K-8 grown in the wastewater of the distillery, under light exposure increased from 3.8 % to 39.8 % of cells dry weight from the seventh to third day of growth during 30 days of cultivation and was 2 times higher the glycogen content of cells on GSB medium. It is assumed that the bacteria C. limicola IMV K-8 use available in the water sources of carbon and other compounds necessary for cell metabolism along with glycogen biosynthesis and bioremediation of wastewater. During C. limicola IMV K-8 growth in the darkness there is an assimilation of organic sources of carbon (acetate, pyruvate and probably organic compounds of wastewater), which allows cells to remain viable for 30 days without additional sources of carbon, nitrogen, etc., but significant glycogen synthesis does not occur. The glycogen formed under phototrophic conditions can be further a source of carbon or a substrate for electric current generation by exoelectrogenic bacteria.

Draft Genome Sequence of Lampenflora Chlorobium limicola Strain Frasassi in a Sulfidic Cave System

The draft genome sequence of Chlorobium limicola strain Frasassi was assembled from metagenomic sequencing of a green mat in an artificially lighted aquarium inside the Frasassi caves in Italy. The genome is 2.08 Mbp in size and contains the necessary genes for anoxygenic photosynthesis and CO 2 fixation.

The processes of lipid peroxidation in the cells of Chlorobium limicola IMV K-8 under the influence of copper (II) sulphate

The effect of stressors, including heavy metal ions such as Cu2+, promotes activation of free radical processes in the cells of microorganisms, which causes changes in their physiological and biochemical properties and the structure of bacterial membranes. The aim of this work was to assess the influence of copper (II) sulphate on intensity of lipid peroxidation (LPO) of Chlorobium limicola IMV K-8 by measuring the content of primary (conjugated dienes and lipid hydroperoxides) and secondary lipid peroxidation products (TBA-reactive products). Microorganisms were cultivated at a temperature of 28 °C in GSB cultivation medium with exposure to light of wavelength 700–800 nm and intensity 40 lux. A suspension of C. limicola ІМV К-8 cells in the phase of exponential growth was treated for one hour with metal salt solution in concentrations 0.05–0.50 mM for investigation of the influence of copper (II) sulphate on its physiological and biochemical properties. The control samples did not contain any copper (II) sulphate. Biomass was determined by turbidity of diluted cell suspension by application of photoelectric colorimeter KFK-3. A mixture of n-heptane and isopropyl alcohol was added into cell-free extract for conjugated dienes determination. The samples were incubated at room temperature and centrifuged. Water was added into the supernatant and the samples were stirred. Ethanol was added to the heptanes phase and adsorption was measured at 233 nm. The content of lipid hydroperoxides was determined by a method based on protein precipitation by trichloroacetic acid followed by addition of ammonium thiocyanate. The concentration of TBA-reactive products in the cell-free extracts was determined by color reaction with malondialdehyde and thiobarbituric acid exposed to high temperature and acidity of the medium, which causes formation of trimetinic adduct with maximal absorption at 532 nm. It was shown that when CuSO4 was added to the incubation medium the content of conjugated dienes and lipid hydroperoxides increased with the enhancement of salt concentration. However, its value decreased by the seventh and eighth days of cultivation. The content of TBA-reactive products under the influence of copper (II) sulphate varied depending on the duration of cultivation and concentration of the metal. Its highest quantity was observed on the eighth day of cultivation. Thus it was determined that under the influence of CuSO4 the content of conjugated dienes, lipid hydroperoxides and TBA-reactive products increases. This indicates the increased activity of lipid peroxidation processes and the free radical chain reaction damage mechanism to lipids under these conditions.

Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases

Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%–34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in ‘canonical’ mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2–PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases.