Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

Photobiohydrogen Production and Strategies for H2 Yield Improvements in Cyanobacteria.

Hydrogen gas (H2) is one of the potential future sustainable and clean energy carriers that may substitute the use of fossil resources including fuels since it has a high energy content (heating value of 141.65 MJ/kg) when compared to traditional hydrocarbon fuels [1]. Water is a primary product of combustion being a most significant advantage of H2 being environmentally friendly with the capacity to reduce global greenhouse gas emissions. H2 is used in various applications. It generates electricity in fuel cells, including applications in transportation, and can be applied as fuel in rocket engines [2]. Moreover, H2 is an important gas and raw material in many industrial applications. However, the high cost of the H2 production processes requiring the use of other energy sources is a significant disadvantage. At present, H2 can be prepared in many conventional ways, such as steam reforming, electrolysis, and biohydrogen production processes. Steam reforming uses high-temperature steam to produce hydrogen gas from fossil resources including natural gas. Electrolysis is an electrolytic process to decompose water molecules into O2 and H2. However, both these two methods are energy-intensive and producing hydrogen from natural gas, which is mostly methane (CH4) and in steam reforming generates CO2 and pollutants as by-products. On the other hand, biological hydrogen production is more environmentally sustainable and less energy intensive than thermochemical and electrochemical processes [3], but most concepts are not yet developed to production scale.

Highly effective reduction of phosphate and harmful bacterial community in shrimp wastewater using short-term biological treatment with immobilized engineering microalgae.

Shrimp farming wastewater includes high amounts of phosphate and microbiological contaminants, necessitating further treatment before release into receiving water bodies. After 24 h of shrimp wastewater treatment, alginate beads containing the blue-green algal Synechocystis strain lacking the phosphate regulator gene (mutant strain ΔSphU) at 150 mg L-1 reduced phosphate content from 17.5 mg L-1 to 5.0 mg L-1, representing 71.5% removal efficiency, with phosphate removal rate reaching 6.9 mg gDW-1 h-1 during photobioreactor operation. For short-term treatment, removal rates of nitrate, ammonium and nitrite were 42.7, 48.5 and 92.9%, respectively. Microalgal encapsulated beads also impacted the bacterial community composition dynamics in shrimp wastewater. Next-generation sequencing targeting the V3-V4 region of the 16S rDNA gene showed significant differences in bacterial community composition after 24 h of treatment. Proteobacteria are the most abundant phylum in shrimp wastewater. After 24 h of bioremediation, reductions of harmful bacteria in the Cellvibrionaceae and Pseudomonadaceae families were recorded at 5.85 and 3.18%, respectively. Engineered microalgal immobilization under optimal conditions can be applied as an alternative short-term bioremediation strategy to remove phosphate and other harmful microbial contamination from shrimp farming wastewater.

Effects of high-strength landfill leachate effluent on stress-induced microalgae lipid production and post-treatment micropollutant degradation.

This research investigates the effects of landfill leachate effluent concentrations from moving bed biofilm reactor (MBBR) on stress-induced Chlorella vulgaris and Scenedesmus armatus lipid production and post-treatment micropollutant degradation. The effluent concentrations were varied between 25%, 50%, 75%, and 100% (v/v). The landfill leachate influent was treated using two-stage moving bed biofilm reactor under 24 h and 18 h hydraulic retention time (HRT). The results indicated that the effluent concentration was positively correlated with the stress-induced microalgae lipid production in the post-treatment of residual micropollutants. C. vulgaris and S. armatus completely remove residual micropollutants in the effluent. The superoxide dismutase and peroxidase activity were positively correlated with the cellular lipid content. The lipid content of C. vulgaris and S. armatus cultivated in the 18 h HRT effluent were 31-51% and 51-64%, while those in the 24 h HRT effluent were 15-16% and 5-19%. The optimal condition of microalgae cultivation for the post-treatment of residual micropollutants was 50-75% (v/v) effluent concentrations under 18 h HRT, achieving the highest lipid production of 113-116 mg/L for C. vulgaris and 74-75 mg/L for S. armatus. Essentially, the MBBR landfill leachate effluent holds promising potential as a substrate for microalgae lipid production.

Conversion of golden oyster mushroom, Pleurotus citrinopileatus to sugar syrup using enzymatic hydrolysis as a substrate for novel bacterial cellulose (Nata) fermentation.

Enzymatic hydrolysis of the golden oyster mushroom (Pleurotus citrinopileatus) generated a new bacterial cellulose (BC). The sugar syrup obtained from the hydrolysis of mushroom powder by commercial enzymes gave maximum total soluble solids (TSS) content at 8.83 ± 0.29°Brix, while 8.82 ± 0.06 mg GAE/g substrate of total phenolic content (TPC) was obtained when using initial substrate and enzyme concentrations at 125 g/L and 5.0%, respectively. Glutamic acid, aspartic acid, alanine and valine were determined as the main amino acids found in P. citrinopileatus hydrolysis at 524.74 ± 0.03, 247.09 ± 0.04, 176.82 ± 0.07 and 174.57 ± 0.01 mg/100 g sample, respectively. Thin-layer chromatography revealed that the obtained sugar syrup was glucose. The hydrolyzed mushroom fermented with Komagataeibacter xylinus AGR 60 at 30 ± 2 °C for 9 days produced optimal conditions at 4.0°Brix of the initial mushroom syrup and 12.0% (v/v) of the starter culture. Maximum BC thickness was 0.88 ± 0.03 cm with 7.90 ± 0.07 g dry weight, equivalent to 39.50 ± 0.35 g/L and 4.39 ± 0.04 g/L/day for BC production (P) and BC production rate (R p), respectively. The obtained BC was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, small-angle X-ray scattering and wide-angle X-ray diffraction. These showed the structure and functional properties as a natural source of fiber from the fermentation of a novel substrate.

Utilization of shrimp wastewater for poly-ß-hydroxybutyrate production by Synechocystis sp. PCC 6803 strain ΔSphU cultivated in photobioreactor.

The wastewater discharge from the intensive shrimp aquaculture contains high concentration of nutrients, which can lead to eutrophication. This study aimed to reuse the shrimp wastewater for low cost cyanobacterial cultivation to produce biodegradable plastic poly-ß-hydroxybutyrate (PHB). The Synechocystis sp. PCC 6803 (ΔSphU) lacking phosphate regulator (SphU) could utilize nutrients in shrimp wastewater for promoting biomass yield of 500 mg L-1 after 14 days. The ΔSphU showed the highest phosphate uptake rate of 20.16 mggDw-1d-1 at the first day of photobioreactor running. In addition, the nutrient removal efficiencies were 96.99% for phosphate, 80.10% for nitrate, 67.90% for nitrite and 98.07% for ammonium. The reduction of nitrate in shrimp wastewater due to nitrogen assimilation could induce PHB accumulation in ΔSphU. The highest PHB content was 32.48% (w/w) DW, with the maximum PHB productivity of 12.73 mg L-1d-1. The produced PHB of ΔSphU had material properties similar to those of the commercial PHB.

Microalgal hydrogen production - A review.

Bio-hydrogen from microalgae including cyanobacteria has attracted commercial awareness due to its potential as an alternative, reliable and renewable energy source. Photosynthetic hydrogen production from microalgae can be interesting and promising options for clean energy. Advances in hydrogen-fuel-cell technology may attest an eco-friendly way of biofuel production, since, the use of H2 to generate electricity releases only water as a by-product. Progress in genetic/metabolic engineering may significantly enhance the photobiological hydrogen production from microalgae. Manipulation of competing metabolic pathways by modulating the certain key enzymes such as hydrogenase and nitrogenase may enhance the evolution of H2 from photoautotrophic cells. Moreover, biological H2 production at low operating costs is requisite for economic viability. Several photobioreactors have been developed for large-scale biomass and hydrogen production. This review highlights the recent technological progress, enzymes involved and genetic as well as metabolic engineering approaches towards sustainable hydrogen production from microalgae.

Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes.

Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3-hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate.

GABA accumulation in response to different nitrogenous compounds in unicellular cyanobacterium Synechocystis sp. PCC 6803.

GABA accumulation and glutamate decarboxylase (GAD) activity, the principal enzyme involved in GABA formation, was investigated in cyanobacterium Synechocystis sp. PCC 6803 wild-type (WT) and gad knockout mutant strains grown in medium containing different nitrogenous compounds. Nitrate was the best nitrogen source for GAD activity and GABA accumulation followed by nitrite, ammonium, and urea. An increase in the accumulation of GABA was observed in WT and mutant cells grown for 24 h in medium supplemented with 0.5 mM putrescine or spermidine with a parallel increase in GAD activity. The mutant could not accumulate GABA at all the conditions tested except when supplemented with putrescine or spermidine, where high GABA levels were observed in both WT and mutant strains. Glutamate supplementation up to 10 mM for 24 h resulted in a significant increase in both GAD activity and GABA content. Overall results suggested that optimization of nitrogen source and nitrogenous compounds supplementation was effective for the enhancement of GABA accumulation in Synechocystis.

Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium Anabaena siamensis TISTR 8012.

BACKGROUND: Biohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase. RESULTS: An engineered strain lacking a functional uptake hydrogenase (∆hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the ∆hupS strain showed long term, sustained hydrogen production under light exposure with 2-3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ∆hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ∆hupS strain. CONCLUSIONS: Inactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

Redirecting the electron flow towards the nitrogenase and bidirectional Hox-hydrogenase by using specific inhibitors results in enhanced H2 production in the cyanobacterium Anabaena siamensis TISTR 8012.

The inhibition of competitive metabolic pathways by various inhibitors in order to redirect electron flow towards nitrogenase and bidirectional Hox-hydrogenase was investigated in Anabaena siamensis TISTR 8012. Cells grown in BG11(0) supplemented with KCN, rotenone, DCMU, and DL-glyceraldehyde under light condition for 24 h showed enhanced H(2) production. Cells grown in BG11 medium showed only marginal H(2) production and its production was hardly increased by the inhibitors tested. H(2) production with either 20mM KCN or 50 µM DCMU in BG11(0) medium was 22 µmol H(2) mg chl a(-1) h(-1), threefold higher than the control. The increased H(2) production caused by inhibitors was consistent with the increase in the respective Hox-hydrogenase activities and nifD transcript levels, as well as the decrease in hupL transcript levels. The results suggested that interruption of metabolic pathways essential for growth could redirect electrons flow towards nitrogenase and bidirectional Hox-hydrogenase resulting in increased H(2) production.

Increased H2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway.

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H(2) production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (ΔnarB) or nitrite reductase (ΔnirA) or both nitrate reductase and nitrite reductase (ΔnarB:ΔnirA) were constructed and tested for their ability to produce hydrogen. H(2) production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H(2) production was observed in the ΔnarB:ΔnirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H(2) production. The results suggest that the high rate of H(2) production observed in the ΔnarB:ΔnirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.