Effect of Glycerol Concentration and Light Intensity on Growth and Biochemical Composition of Arthrospira (Spirulina) Platensis: A Study in Semi-Continuous Mode with Non-Aseptic Conditions

In this study, Arthrospira platensis was grown in the presence of different glycerol concentrations (0.5–9 g/L) under three light intensities (5, 10 and 15 Klux) in semi-continuous mode and under non-axenic conditions. The aim of this study was to investigate the growth performance, the biomass biochemical composition and any interactions between A. platensis and bacteria that would potentially grow as well on glycerol. The results here show that glycerol did not have any positive effect on biomass production of A. platensis. In contrast, it was observed that by increasing glycerol concentration the growth performance of A. platensis was restricted, while a gradual increase of bacteria population was observed, which apparently outcompeted and repressed A. platensis growth. Chlorophyll fluorescence measurements (Quantum Yields) revealed that glycerol was not an inhibiting factor per se of photosynthesis. On the other hand, cyanobacterial biomass grown on glycerol displayed a higher content in proteins and lipids. Especially, protein productivity was enhanced around 15–35% with the addition of glycerol compared to the control. In distinction, carbohydrate and photosynthetic pigments (phycocyanin and chlorophyll-α) content decreased with the increase of glycerol concentration. The results here suggest that A. platensis did not utilize glycerol for biomass production but most probably as metabolic energy carrier towards synthesis of proteins and lipids, which are more energy consuming metabolites compared to carbohydrates. The study revealed that the addition of glycerol at amounts of 0.5–1.5 g/L could be a strategy to improve protein productivity by A. platensis

Continuous Culture of <i>Auxenochlorella protothecoides</i> on Biodiesel Derived Glycerol under Mixotrophic and Heterotrophic Conditions: Growth Parameters and Biochemical Composition

As crude glycerol comprises a potential substrate for microalga fermentation and value added products’ biosynthesis, Auxenochlorella protothecoides was grown on it under heterotrophic and mixotrophic conditions and its growth kinetics were evaluated in a continuous system under steady state conditions. Increasing initial glycerol concentration (from 30 to 50 g/L) in the heterotrophic culture led to reduced biomass yield (Yx/S) and productivity (Px), but favored lipid accumulation. Under heterotrophic conditions, the microalga was found to grow better (biomass up to 7.888 g/L) and faster (higher growth rates), the system functioned more effectively (higher Px) and crude glycerol was exploited more efficiently. Heterotrophy also favored proteins synthesis (up to 53%), lipids (up to 9.8%), and carbohydrates (up to 44.6%) accumulation. However, different trophic modes had no significant impact on the consistency of proteins and lipids. Oleic acid was the most abundant fatty acid detected (55–61.2% of the total lipids). The algal biomass contained many essential and non-essential amino acids, especially arginine, glutamic acid, lysine, aspartic acid, leucine, and alanine. In all the experimental trials, the protein contents in the microalgal biomass increased with the increasing dilution rate (D), with a concomitant decrease in the lipids and carbohydrates fractions

Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation▿

Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3− produced by CAA1

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic <i>Pseudomonas stutzeri</i> and <i>Pseudomonas</i> sp. Isolated from Distal and Close Geographical Regions

<div><p>The presence of nitrogen fixers within the genus <i>Pseudomonas</i> has been established and so far most isolated strains are phylogenetically affiliated to <i>Pseudomonas stutzeri</i>. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic <i>P. stutzeri</i> strains and <i>Pseudomonas azotifigens</i> revealed that all are flanked by genes coding for cobalamin synthase (<i>cobS</i>) and glutathione peroxidise (<i>gshP</i>). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis <i>P. stutzeri</i> DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other <i>P. stutzeri</i> strains carry only one set of repeats. The genetic diversity of eleven diazotrophic <i>Pseudomonas</i> isolates was also investigated. Multilocus sequence typing grouped nine isolates along with <i>P. stutzeri</i> and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic <i>Pseudomonas</i> isolates is flanked by <i>cobS</i> and <i>gshP</i> genes. Furthermore, we demonstrated that the putative NFI of <i>Pseudomonas</i> sp. Gr65 is flanked by inverted repeats identical to those found in <i>P. stutzeri</i> DSM 4166 and while the other <i>P. stutzeri</i> isolates harbor the repeats located in the intergenic region between <i>cobS</i> and glutaredoxin genes as in the case of <i>P. stutzeri</i> A1501. Taken together these data suggest that all putative NFIs of diazotrophic <i>Pseudomonas</i> isolates are anchored in an intergenic region between <i>cobS</i> and <i>gshP</i> genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic <i>Pseudomonas</i> strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.</p></div

Inverted and/or direct repeats identified in the IRLeft and IRRight regions flanking the nitrogen fixation island of <i>P. stutzeri</i> strains and <i>Pseudomonas</i> sp. Gr65.

<p>The repeats DR1, DR2 and DR3 (boxed) present in the IRLeft and IRRight regions flanking the nitrogen fixation island of <i>P. stutzeri</i> A1501 and DSM4166 (A), <i>P. stutzeri</i> NF13 (B) and <i>Pseudomonas</i> sp. Gr65 (D). The repeats located in the intergenic region between PST_1322- PST_1323 (designated as 1501Μ) and PSTAA_1354- PSTAA_1355 (designated as 4166Μ) (C). The coordinates displayed on the left and the right side of the sequences indicate the position of the sequences in genome of <i>P. stutzeri</i> A1501 or DSM4166 (A and C). The coordinates displayed for <i>Pseudomonas</i> sp. Gr65 were based on the nucleotide sequences of IRLeft and IRRight found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105837#pone.0105837.s002" target="_blank">files S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105837#pone.0105837.s003" target="_blank">S2</a>.</p

16S rRNA phylogenetic tree.

<p>Neighbor-Joining phylogenetic tree of 16S rRNA gene constructed using the partial nucleotide sequence from the 11 <i>P. stutzeri</i> isolates and related sequences obtained from NCBI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105837#pone.0105837-Anzai1" target="_blank">[40]</a>. Numbers shown at nodes indicate bootstrap values (percentage of 1000 replicates). The trees were constructed by the neighbour-joining method using MEGA v.5. Reference strains are highlighted in bold. The bar scale indicates the rates of substitution per nucleotide position. Sequence accession numbers are given in parentheses. ^T = type strain.</p

Rep-PCR genomic fingerprinting of <i>P. stutzeri</i> strains.

<p>Rep-PCR genomic fingerprinting of <i>P. stutzeri</i> A1501, <i>P. stutzeri</i> DSM4611 and 11 isolates (Gr16, Gr17, Gr18, Gr19, Gr20, Gr50, Gr45, Gr46, Gr57, Gr65). M: DNA ladder λ DNA HindIII and φX174 DNA HaeIII.</p

Schematic representation and comparison of the Nitrogen Fixation Islands and flanking genes of diazotrophic <i>P. stutzeri</i> strains A1501, DSM4166, B1SMN1, KOS6 and <i>P. azotifigens</i> DSM 17556.

<p>The nitrogen fixation island of <i>P. stutzeri</i> KOS6 was assembled downloading from the Integrated Microbial Genomes (IMG) (<a href="https://img.jgi.doe.gov/cgi-bin/w/main.cgi" target="_blank">https://img.jgi.doe.gov/cgi-bin/w/main.cgi</a>) three contigs (AMCZ01000041, AMCZ01000045 and AMCZ000005) indicated by brackets. The nitrogen fixation island of <i>P. stutzeri</i> strains B1SMN1 and <i>P. azotifigens</i> DSM 17556 were found in one contig. The colored of arrows are indicating different functional genes as described by IMG.</p