Erythromycin mediates co-flocculation between cyanobacterium Synechocystis sp. PCC 6803 and filamentous fungi in liquid cultivation without organic compounds.

Photoautotrophic cyanobacteria assimilate the greenhouse gas carbon dioxide as their sole carbon source for producing useful bioproducts. However, harvesting the cells from their liquid media is a major bottleneck in the process. Thus, an easy-to-harvest method, such as auto-flocculation, is desirable. Here, we found that cyanobacterium Synechocystis sp. PCC 6803 co-flocculated with a natural fungal contamination in the presence of the antibiotic erythromycin (EM) but not without EM. The fungi in the co-flocculated biomass were isolated and found to consist of five species with the filamentous Purpureocillium lilacinum and Aspergillus protuberus making up 71% of the overall fungal population. The optimal co-cultivation for flocculation was an initial 5 mg (fresh weight) of fungi, an initial cell density of Synechocystis of 0.2 OD730, 10 µM EM, and 14 days of cultivation in 100 mL of BG11 medium with no organic compound. This yielded 248 ± 28 mg/L of the Synechocystis-fungi flocculated biomass from 560 ± 35 mg/L of total biomass, a 44 ± 2% biomass flocculation efficiency. Furthermore, the EM treated Synechocystis cells in the Synechocystis-fungi flocculate had a normal cell color and morphology, while those in the axenic suspension exhibited strong chlorosis. Thus, the occurrence of the Synechocystis-fungi flocculation was mediated by EM, and the co-flocculation with the fungi protected Synechocystis against the development of chlorosis. Transcriptomic analysis suggested that the EM-mediated co-flocculation was a result of down-regulation of the minor pilin genes and up-regulation of several genes including the chaperone gene for pilin regulation, the S-layer protein genes, the exopolysaccharide-polymerization gene, and the genes for signaling proteins involved in cell attachment and abiotic-stress responses. The CuSO4 stress can also mediate Synechocystis-fungi flocculation but at a lower flocculation efficiency than that caused by EM. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.

Microalgal-bacterial immobilized co-culture as living biofilters for nutrient recovery from synthetic wastewater and their potential as biofertilizers.

This study investigates nutrient recovery from synthetic municipal wastewater using co-immobilized cultures of Chlorella vulgaris TISTR 8580 (CV) and plant growth-promoting bacteria, Bacillus subtilis TISTR 1415 (BS) as living biofilters for a subsequent biofertilizer activity. The optimal condition for nutrient recovery was at the 1:1 ratio of CV/BS using mixed guar gum/carrageenan (GG/CG) binders. After 7-day wastewater treatment, the living biofilters removed 86.7 ± 0.5% of ammonium and 99.3 ± 0.3% of phosphates and were tested subsequently as biofertilizers for 20 days to grow selected plants. The highest optimal biomass and chlorophyll a content was 2 ± 0.3 g (CV/BS 3:1) and 12.4 ± 0.7 µg/g (CV/BS 1:1) from cucumber respectively, however, the close-to-neutral pH (8.0 ± 0.3) was observed from sunflower using CV/BS 1:1 living biofilters. Conclusively, the designed living biofilters exhibit the potential to recover nutrients from wastewater and be used as biofertilizers for circular agriculture.

Determination of Poly(3-hydroxybutyrate) Content in Cyanobacterium Synechocystis sp. PCC 6803 Using Acid Hydrolysis Followed by High-performance Liquid Chromatography.

Various photoautotrophic cyanobacteria accumulate intracellular poly(3-hydroxybutyrate) (PHB) granules. This protocol can be used for determining the PHB contents of the cells as % PHB weight per dry cell weight using acid hydrolysis followed by high-performance liquid chromatography (HPLC). This HPLC analysis is rapid, with a running time of approximately 5 min per sample. The technique can accurately determine PHB concentrations in the range of 2-1,000 µg/mL PHB. However, this technique is not applicable for determining the contents of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in cyanobacteria.

Chemical Triggering Cyanobacterial Glycogen Accumulation: Methyl Viologen Treatment Increases Synechocystis sp. PCC 6803 Glycogen Storage by Enhancing Levels of Gene Transcript and Substrates in Glycogen Synthesis.

Two-stage cultivation is effective for glycogen production by cyanobacteria. Cells were first grown under adequate nitrate supply (BG11) to increase biomass and subsequently transferred to nitrogen deprivation (-N) to stimulate glycogen accumulation. However, the two-stage method is time-consuming and requires extensive energy. Thus, one-stage cultivation that enables both cell growth and glycogen accumulation is advantageous. Such one-stage method could be achieved using a chemical triggering glycogen storage. However, there is a limited study on such chemicals. Here, nine compounds previously reported to affect cyanobacterial cellular functions were examined in Synechocystis sp. PCC 6803. 2-Phenylethanol, phenoxyethanol, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and methyl viologen can stimulate glycogen accumulation. The oxidative stress agent, methyl viologen significantly increased glycogen levels up to 57% and 69% [w/w dry weight (DW)] under BG11 and -N cultivation, respectively. One-stage cultivation where methyl viologen was directly added to the pre-grown culture enhanced glycogen storage to 53% (w/w DW), compared to the 10% (w/w DW) glycogen level of the control cells without methyl viologen. Methyl viologen treatment reduced the contents of total proteins (including phycobiliproteins) but caused increased transcript levels of glycogen synthetic genes and elevated levels of metabolite substrates for glycogen synthesis. Metabolomic results suggested that upon methyl viologen treatment, proteins degraded to amino acids, some of which could be used as a carbon source for glycogen synthesis. Results of oxygen evolution and metabolomic analysis suggested that photosynthesis and carbon fixation were not completely inhibited upon methyl viologen treatment, and these two processes may partially generate upstream metabolites required for glycogen synthesis.

Advancement on mixed microalgal-bacterial cultivation systems for nitrogen and phosphorus recoveries from wastewater to promote sustainable bioeconomy.

Biological wastewater treatment is a promising and environmentally friendly method that utilises living microorganisms to remediate water and enable recovery or conversion of contaminants into valuable products. For many decades, microalgae and cyanobacteria, photosynthetic living microorganisms, have been explored extensively for wastewater bioremediation. They can be used for recovering valuable nutrients such as nitrogen and phosphorous from secondary effluents and capable of transforming those nutrients into marketable products such as biofuels, biofertilisers, nutraceutical, and pigments for promoting a Bio-Circular Green economy. In recent years, there has been a shift towards mixing compatible microalgae with bacteria, which is inspired by their natural symbiotic relationships to increase nitrogen and phosphorus recoveries. With this enhanced bioremediation, recovery of polluted wastes can be intensified and higher biomass quality (with high nutrient density) can be achieved. This review focuses on the state-of-the-art of mixed microalgal-bacterial cultivating systems. A comprehensive comparison of existing studies that used Chlorella species as microalgae in various mixed microalgal-bacterial cultivating systems (suspension, biofilm, and immobilisation) for nitrogen and phosphorus recoveries from wastewater is conducted. Key technical challenges such as balancing microalgae and bacteria species, pH regulation, light distribution, biomass harvesting, and biomass conversion are also discussed. From the data comparisons among different cultivation systems, it has been suggested that immobilisation appears to require less amount of operational light compared to the suspended and biofilm-based systems for similar nitrogen and phosphorus removal efficiencies.

Enhanced polyglucan contents in divergent cyanobacteria under nutrient-deprived photoautotrophy: transcriptional and metabolic changes in response to increased glycogen accumulation in nitrogen-deprived Synechocystis sp. PCC 6803.

Cyanobacteria accumulate polyglucan as main carbohydrate storage. Here, the cellular polyglucan content was determined in 27 cyanobacterial strains from 25 genera. The polyglucan contents were significantly enhanced in 20 and 23 strains under nitrogen (-N) and phosphate (-P) deprivation, respectively. High polyglucan accumulation was not associated with particular evolutionary groups but was strain specific. The highest polyglucan accumulations of 46.2% and 52.5% (w/w dry weight; DW) were obtained under -N in Synechocystis sp. PCC 6803 (hereafter Synechocystis) and Chroococcus limneticus, respectively. In Synechocystis, 80-97% (w/w) of the polyglucan was glycogen. Transcriptome and metabolome analyses during glycogen accumulation under -N were determined in Synechocystis. The genes responsible for the supply of the substrates for glycogen synthesis: glycerate-1,3-phosphate and fructose-1,6-phosphate, were significantly up-regulated. The genes encoding the enzymes converting succinate to malate in TCA cycle, were significantly down-regulated. The genes encoding the regulator proteins which inhibits metabolism at lower part of glycolysis pathway, were also significantly up-regulated. The transcript levels of PII protein and the level of 2-oxoglutarate, which form a complex that inhibits lower part of glycolysis pathway, were significantly increased. Thus, the increased Synechocystis glycogen accumulation under -N was likely to be mediated by the increased supply of glycogen synthesis substrates and metabolic inhibitions at lower part of glycolysis pathway and TCA cycle.

Chemicals Affecting Cyanobacterial Poly(3-hydroxybutyrate) Accumulation: 2-Phenylethanol Treatment Combined with Nitrogen Deprivation Synergistically Enhanced Poly(3-hydroxybutyrate) Storage in Synechocystis sp. PCC6803 and Anabaena sp. TISTR8076.

Various photoautotrophic cyanobacteria increase the accumulation of bioplastic poly(3-hydroxybutyrate) (PHB) under nitrogen deprivation (-N) for energy storage. Several metabolic engineering enhanced cyanobacterial PHB accumulation, but these strategies are not applicable in non-gene-transformable strains. Alternatively, stimulating PHB levels by chemical exposure is desirable because it might be applied to various cyanobacterial strains. However, the study of such chemicals is still limited. Here, 19 compounds previously reported to affect bacterial cellular processes were evaluated for their effect on PHB accumulation in Synechocystis sp. PCC6803, where 3-(3,4-dichlorophenyl)-1,1-dimethylurea, methyl viologen, arsenite, phenoxyethanol and 2-phenylethanol were found to increase PHB accumulation. When cultivated with optimal nitrate supply, Synechocystis contained less than 0.5% [w/w dry weight (DW)] PHB, while cultivation under -N conditions increased the PHB content to 7% (w/w DW). Interestingly, the -N cultivation combined with 2-phenylethanol exposure reduced the Synechocystis protein content by 27% (w/w DW) but significantly increased PHB levels up to 33% (w/w DW), the highest ever reported photoautotrophic cyanobacterial PHB accumulation in a wild-type strain. Results from transcriptomic and metabolomic analysis suggested that under 2-phenylethanol treatment, Synechocystis proteins were degraded to amino acids, which might be subsequently utilized as the source of carbon and energy for PHB biosynthesis. 2-Phenylethanol treatment also increased the levels of metabolites required for Synechocystis PHB synthesis (acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA and NADPH). Additionally, under -N, the exposure to phenoxyethanol and 2-phenylethanol increased the PHB levels of Anabaena sp. from 0.4% to 4.1% and 6.6% (w/w DW), respectively. The chemicals identified in this study might be applicable for enhancing PHB accumulation in other cyanobacteria.

Disruption of Hydrogen Gas Synthesis Enhances the Cellular Levels of NAD(P)H, Glycogen, Poly(3-hydroxybutyrate) and Photosynthetic Pigments Under Specific Nutrient Condition(s) in Cyanobacterium Synechocystis sp. PCC 6803.

In photoautotrophic Synechocystis sp. PCC 6803, NADPH is generated from photosynthesis and utilized in various metabolism, including the biosynthesis of glyceraldehyde 3-phosphate (the upstream substrate for carbon metabolism), poly(3-hydroxybutyrate) (PHB), photosynthetic pigments, and hydrogen gas (H2). Redirecting NADPH flow from one biosynthesis pathway to another has yet to be studied. Synechocystis's H2 synthesis, one of the pathways consuming NAD(P)H, was disrupted by the inactivation of hoxY and hoxH genes encoding the two catalytic subunits of hydrogenase. Such inactivation with a complete disruption of H2 synthesis led to 1.4-, 1.9-, and 2.1-fold increased cellular NAD(P)H levels when cells were cultured in normal medium (BG11), the medium without nitrate (-N), and the medium without phosphate (-P), respectively. After 49-52 d of cultivation in BG11 (when the nitrogen source in the media was depleted), the cells with disrupted H2 synthesis had 1.3-fold increased glycogen level compared to wild type of 83-85% (w/w dry weight), the highest level reported for cyanobacterial glycogen. The increased glycogen content observed by transmission electron microscopy was correlated with the increased levels of glucose 6-phosphate and glucose 1-phosphate, the two substrates in glycogen synthesis. Disrupted H2 synthesis also enhanced PHB accumulation up to 1.4-fold under -P and 1.6-fold under -N and increased levels of photosynthetic pigments (chlorophyll a, phycocyanin, and allophycocyanin) by 1.3- to 1.5-fold under BG11. Thus, disrupted H2 synthesis increased levels of NAD(P)H, which may be utilized for the biosynthesis of glycogen, PHB, and pigments. This strategy might be applicable for enhancing other biosynthetic pathways that utilize NAD(P)H.

Disruption of cyanobacterial γ-aminobutyric acid shunt pathway reduces metabolites levels in tricarboxylic acid cycle, but enhances pyruvate and poly(3-hydroxybutyrate) accumulation.

The photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 assimilates carbon dioxide as the sole carbon source, and a major portion of the assimilated carbon is metabolically consumed by the tricarboxylic acid (TCA) cycle. Effects of partial interference of TCA cycle metabolic activity on other carbon metabolism have yet to be examined. Here, the γ-aminobutyric acid (GABA) shunt, one of the metabolic pathways for completing TCA cycle in Synechocystis, was disrupted via inactivating the glutamate decarboxylase gene (gdc). Under normal photoautotrophic condition, cell growth and the level of the TCA cycle metabolites succinate, malate and citrate were decreased by 25%, 35%, 19% and 28%, respectively, in Δgdc mutant relative to those in the wild type (WT). The cellular levels of glycogen and total lipids of the Δgdc mutant were comparable to those of the WT, but the intracellular levels of pyruvate and bioplastic poly(3-hydroxybutyrate) (PHB) were 1.23- and 2.50-fold higher, respectively, in Δgdc mutant. Thus, disruption of the GABA shunt pathway reduced the TCA cycle metabolites levels, but positively enhanced the bioaccumulation of pyruvate and PHB. The PHB production rate in Δgdc mutant was 2.0-fold higher than in the WT under normal photoautotrophy.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) under photoautotrophy and heterotrophy by non-heterocystous N2-fixing cyanobacterium.

The photoautotrophically grown cyanobacterium Oscillatoria okeni TISTR 8549 was found to produce bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This PHBV production occurred under nitrogen deprivation (-N) that yielded PHBV accumulation of 14±4% (w/w DW) in which 3-hydroxyvalerate accounted for 5.5mol%. The heterotrophically grown (-N condition with acetate supplementation) cells under light showed no increase of PHBV storage, but under dark condition these cells increased PHBV accumulation to 42±8% (w/w DW) with 6.5mol% of 3-hydroxyvalerate. Compared to poly-3-hydroxybutyrate (PHB), the PHBV from O. okeni had a lower melting temperature by 5-7°C, a higher % elongation at break by 4-7times and a greater Young's elastic modulus by 2.3-2.5times.

Two-stage (photoautotrophy and heterotrophy) cultivation enables efficient production of bioplastic poly-3-hydroxybutyrate in auto-sedimenting cyanobacterium.

Sustainable production of bioplastics by heterotrophic microbes has been restricted by the limited resources of organic substrates and the energy required for biomass harvest. Here, the easy-to-harvest cyanobacterium (Chlorogloea fritschii TISTR 8527), from which the biomass instantaneously settled to the bottom of liquid culture, was utilized to produce poly-3-hydroxybutyrate (PHB) using a two-stage cultivation strategy. The cells were first pre-grown under normal photoautotrophy to increase their biomass and then recultivated under a heterotrophic condition with a single organic substrate to produce the product. Through optimization of this two-stage cultivation, the mass conversion efficiency of acetate substrate to PHB was obtained at 51 ± 7% (w/w), the comparable level to the theoretical biochemical conversion efficiency of acetate to PHB. This two-stage cultivation that efficiently converted the substrate to the product, concurrent with a reduced culture biomass, may be applicable for the production of other biopolymers by cyanobacteria.

Increased accumulation of polyhydroxybutyrate in divergent cyanobacteria under nutrient-deprived photoautotrophy: An efficient conversion of solar energy and carbon dioxide to polyhydroxybutyrate by Calothrix scytonemicola TISTR 8095.

The cellular PHB content was determined in 137 strains of cyanobacteria representing 88 species in 26 genera under six photoautotrophic nutrient conditions. One hundred and thirty-four strains were PHB producers. The PHB contents of these 134 strains were subtle under normal growth condition, but were significantly increased in 63 strains under nitrogen deprivation (-N), a higher frequency than with phosphate and/or potassium and all-nutrient deprivation. A high PHB accumulation was not associated with any particular evolutionary groups, but was strain specific. The filamentous Calothrix scytonemicola TISTR 8095 produced 356.5±63.4mg/L PHB under -N from a biomass of 1396.6±66.1mg/L, giving a PHB content of 25.4±3.5% (w/w dry weight). This PHB productivity is equivalent to the CO2 consumption of 729.2±129.8mg/L. The maximum energy conversion from solar energy to PHB obtained by C. scytonemicola TISTR 8095 was 1.42±0.30%.

Increased bacterial hemolytic activity is conferred by expression of TlyA methyltransferase but not by its 2'-O-methylation of the ribosome.

Bacterial 2'-O-methyltransferase TlyA methylates either both nucleotide C1409 of 16S rRNA and C1920 of 23S rRNA or only the C1920. Both ribosomal methylations increase bacterial susceptibility to ribosome-targeting antibiotics capreomycin and viomycin. However, TlyA has been suggested to also function as a hemolysin. Here, heterologous expression of TlyA from six diverse bacteria (including Mycobacterium tuberculosis and M. smegmatis) was found to increase hemolytic ability in the Escherichia coli host. Characterizing E. coli strains expressing mycobacterial TlyA with mutated rRNA recognition domain and impaired rRNA methylations showed that the abolished C1409 methylation altogether with significantly reduced C1920 methylation did not affect E. coli hemolytic activity. Thus, the increased bacterial hemolytic function is not likely a consequence of TlyA-mediated methylations of the ribosome. Purified water-soluble TlyA showed a weak concentration-dependent hemolysis in vitro. Therefore, the TlyA isoform alone is not a potent hemolysin. The results suggested that the bacterial hemolytic function might relate to the over-expression of TlyA and its interaction to other non-ribosomal target that is associated with the hemolytic ability.

Capreomycin susceptibility is increased by TlyA-directed 2'-O-methylation on both ribosomal subunits.

The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to capreomycin and viomycin. We report here that TlyA orthologues occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyA(I) , has shorter N- and C-termini and methylates only C1920; the second group (now TlyA(II) ) includes the mycobacterial enzyme, and these longer orthologues methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologues. Amino acid substitutions at the N-terminus of TlyA(II) reduce its ability to methylate these substrates. Growing pairs of recombinant TlyA(II) Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of capreomycin. The findings reveal that 2'-O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of capreomycin and viomycin on the bacterial ribosome.

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG.

The RsmG methyltransferase is responsible for N(7) methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 A resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.

Mutations in conserved helix 69 of 23S rRNA of Thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications.

Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of Thermus thermophilus selected for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, DeltamU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2'-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations.

A cyanobacterial strain with all chromosomal rRNA operons inactivated: a single nucleotide mutation of 23S rRNA confers temperature-sensitive phenotypes.

The presence of a multicopy chromosome, with each copy containing two rRNA operons (rrnA and rrnB), has been an obstacle to analysing mutated rRNA in Synechococcus PCC 7942. To create a system for expressing homogeneous mutated rRNA, the chromosomal rrn operons were sequentially inactivated and a final strain was successfully obtained with all the chromosomal rrn operons inactivated but carrying a replaceable multicopy plasmid containing a single rrn operon. The lag time required for growth response on dark/light shift of mutant strains with chromosomal rrnA or rrnB inactivated was increased 50 % over that of the wild-type strain; however, the presence of the plasmid-borne rrn operon restored the lag time. The doubling time of mutant strains carrying only a functional rrnB operon, but not strains carrying only a functional rrnA operon, was significantly longer than that of the wild-type strain. A strain in which essentially all the cellular 23S rRNA contained the mutation C2588A was temperature sensitive at 16 degrees C and 45 degrees C. Position C2588 is equivalent to C2611 of the peptidyltransferase centre in domain V of Escherichia coli 23S rRNA.