Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L⁻¹·day⁻¹, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.     

Analysis of carbohydrate storage granules in the diazotrophic cyanobacterium Cyanothece sp. PCC 7822

The unicellular diazotrophic cyanobacteria of the genus Cyanothece demonstrate oscillations in nitrogenase activity and H₂ production when grown under 12 h light–12 h dark cycles. We established that Cyanothece sp. PCC 7822 allows for the construction of knock-out mutants and our objective was to improve the growth characteristics of this strain and to identify the nature of the intracellular storage granules. We report the physiological and morphological effects of reduction in nitrate and phosphate concentrations in BG-11 media on this strain. We developed a series of BG-11-derived growth media and monitored batch culture growth, nitrogenase activity and nitrogenase-mediated hydrogen production, culture synchronicity, and intracellular storage content. Reduction in NaNO₃ and K₂HPO₄ concentrations from 17.6 and 0.23 to 4.41 and 0.06 mM, respectively, improved growth characteristics such as cell size and uniformity, and enhanced the rate of cell division. Cells grown in this low NP BG-11 were less complex, a parameter that related to the composition of the intracellular storage granules. Cells grown in low NP BG-11 had less polyphosphate, fewer polyhydroxybutyrate granules and many smaller granules became evident. Biochemical analysis and transmission electron microscopy using the histocytochemical PATO technique demonstrated that these small granules contained glycogen. The glycogen levels and the number of granules per cell correlated nicely with a 2.3 to 3.3-fold change from the minimum at L0 to the maximum at D0. The differences in granule morphology and enzymes between Cyanothece ATCC 51142 and Cyanothece PCC 7822 provide insights into the formation of large starch-like granules in some cyanobacteria.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.     

The atp1 and atp2 operons of the cyanobacterium Synechocystis sp. PCC 6803

The two operons atp1 and atp2, encoding the subunits of the F_OF₁ ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis F_OF₁ ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5 end of atp1, coding for a putative gene product similar to uncI in Escherichia coli.A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Q_y transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Q_y transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.Abbreviations APC - allophycocyanin - Chl a - chlorophyll a - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LD - linear dichroism - LD/A - linear dichroism divided by absorbance - LHC - light-harvesting complex - PBS - phycobilisome - PC - phycocyanin - PS - Photosystem     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Nutrient acquisition and limitation for the photoautotrophic growth of Synechocystis sp. PCC6803 as a renewable biomass source

Photoautotrophic microorganisms (cyanobacteria and algae) offer high promise as a source of biomass for renewable energy due to their rapid growth rates and high biomass yields. To provide a framework for evaluating the feasibility of growing phototrophic microorganisms with high biomass production rates, we operated a bench‐scale photobioreactor using Synechocystis sp. PCC6803 and with light conditions imitating actual day–night light irradiance (LI). During the time of peak LI, PCC6803's specific growth rate (1.7 day⁻¹) and the nitrate uptake rate (0.46 g N/g DW day) were high compared to past reports. Analysis employing the stoichiometry of photosynthesis of PCC6803 and ionic speciation showed that bicarbonate and phosphate were driven to very low concentrations for the high‐LI conditions. In particular, the systematic evaluation of rate‐limiting factors identified when the CO₂–C_i supply rate needed to be increased to mitigate HCO depletion and a large pH increase. It also showed that the traditional BG‐11 medium needs to be augmented with phosphate to avoid severe P depletion. This work exploits quantitative understanding the stoichiometry and kinetics of cyanobacteria for the high‐rate production of a renewable biomass. Biotechnol. Bioeng. 2011;108: 277–285. © 2010 Wiley Periodicals, Inc.     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.     

Characterization of two Synechococcus sp. PCC7002‐related cyanobacterial strains in relation to 16S rDNA,crtR gene,lipids and pigments

Two Synechococcus strains from the Culture Collection of the Institute for Marine Sciences of Andalusia (Cádiz, Spain), namely Syn01 and Syn02, were found to be closely related to the model strain Synechococcus sp. PCC7002 according to 16S rDNA (99% identity). Pigment and lipid profiles and crtR genes of these strains were ascertained and compared. The sequences of the crtR genes of these strains were constituted by 888 bp, and showed 99% identity between Syn01 and Syn02, and 94% identity of Syn01 and Syn02 to Synechococcus sp. PCC7002. There was coincidence in photosynthetic pigments between the three strains apart from the pigment synechoxanthin, which could be only observed in Synechococcus sp. PCC7002. Species of sulfoquinovosyl‐diacyl‐glycerol (SQDG), phosphatidyl‐glycerol (PG), mono‐ and di‐galactosyl‐diacyl‐glycerol (MGDG and DGDG) were detected by high performance liquid chromatography‐mass spectrometry analysis of lipid extracts. The most abundant species within each lipid class were those containing C18:3 together with C16:0 fatty acyl substituents in the glycerol backbone of the same molecule. From these results it is concluded that these cyanobacterial strains belong to group 2 of the lipid classification of cyanobacteria.     

Functional analysis of the PsbX protein by deletion of the corresponding gene in Synechocystis sp. PCC 6803

The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.     

Engineering of a green‐light inducible gene expression system in Synechocystis sp. PCC6803

In order to construct a green‐light‐regulated gene expression system for cyanobacteria, we characterized a green‐light sensing system derived from Synechocystis sp. PCC6803, consisting of the green‐light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (P_cpcG2). CcaS and CcaR act as a genetic controller and activate gene expression from P_cpcG2 with green‐light illumination. The green‐light induction level of the native P_cpcG2 was investigated using GFPuv as a reporter gene inserted in a broad‐host‐range vector. A clear induction of protein expression from native P_cpcG2 under green‐light illumination was observed; however, the expression level was very low compared with P_trc, which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine‐Dalgarno‐like sequence derived from the cpcB gene was inserted in the 5′ untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green‐light sensing system resulted in about 40‐fold higher protein expression than with the wild‐type promoter with a high ON/OFF ratio under green‐light illumination. The engineered green‐light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses.     

Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and Nostoc PCC7936

Two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested with regard to their metal removal capability by using copper as model metal. The experiments, carried out with the sole cyanobacterial biomass suspended in distilled water and confined into small dialysis tubings, showed that C. capsulata biomass is characterized by the best efficiency in metal removal, with a q_max (maximum amount of copper removed per biomass unit) of 96 ± 2 mg Cu(II) removed per g of protein in comparison with the value of 79 ± 3 of Nostoc PCC7936 biomass. The experimental data obtained with both cyanobacterial biomass best fit the Langmuir sorption isotherm. The sorption of copper started from the first minutes of contact with the metal and attained the equilibrium state, when no more copper removal was evident, after 5 and 6 hours, for C. capsulata and Nostoc PCC7936, respectively. The best efficiency in Cu(II) removal was obtained at pH 6.1–6.2, while the presence of Mg²⁺ or Ca²⁺ reduced copper removal capability of both species to 60–70% of their q_max. The results showed that the biomass of C. capsulata and Nostoc PCC7936 possesses a high affinity and a high specific uptake for copper, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use the capsulated trichomes of the two cyanobacteria for the bioremoval of heavy metals from polluted water bodies.     

Improvement of carbon dioxide biofixation in a photobioreactor using Anabaena sp. PCC 7120

The biological photosynthetic process is useful and environmentally benign compared with other carbon dioxide (CO₂) mitigation processes. In the present study, Anabaena sp. PCC 7120 was utilized for carbon dioxide mitigation. A customized airlift photobioreactor was found to provide higher light utilization efficiency and a higher rate of CO₂ biofixation compared with that of a bubble column. The maximum biomass concentrations were 0.71 and 1.13 g L^?1 in the bubble column and airlift photobioreactor, respectively, using BG11₀ medium under aerated conditions. A lower mixing time in the airlift photobioreactor compared with that of the bubble column resulted in improved mass transfer. The CO₂ biofixation rate of Anabaena sp. PCC 7120 was determined using different phosphate concentrations at a light intensity of 120 μE m^?2 s^?1 and 5% (v/v) CO₂-enriched air in the airlift photobioreactor. However, it was observed that the specific growth rate was independent at higher light intensity. In addition, it was observed that increased light intensity, phosphate and CO₂ concentrations could enhance the CO₂ biofixation efficiency to a greater extent.     

A conditional expression vector for the cyanobacteriaSynechocystis sp. strains PCC6803 and PCC6714 orSynechococcus sp. strains PCC7942 and PCC6301

An expression vector, pFC1, has been constructed based on the promiscuous plasmid RSF1010, which provides autonomous replication in several cyanobacteria of the generaSynechocystis andSynechococcus [Mermet-Bouvier et al., Curr Microbiol 26:323–327]. pFC1 harbors the cI857 repressor-encoding gene andp _R promoter, followed by the cro ribosome-binding site and ATG translation initiation codon. The latter is located within the uniqueNdeI restriction site (CATATG) of pFC1 and can be exposed after cleavage with this enzyme for in-frame fusion with the protein-coding sequence to be expressed. TheEscherichia coli lacZ reporter gene cloned in pFC1 appeared to be highly expressed in heat-inducedE. coli or cyanobacterial cells. In every case, -galactosidase amounted to at least 10% of soluble proteins.     

Cytochrome c6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119

Most organisms performing oxygenic photosynthesis contain either cytochrome c ₆ or plastocyanin, or both, to transfer electrons from cytochrome b ₆-f to photosystem I. Even though plastocyanin has superseded cytochrome c ₆ along evolution, plants contain a modified cytochrome c ₆, the so called cytochrome c _6A, whose function still remains unknown. In this article, we describe a second cytochrome c ₆ (the so called cytochrome c ₆-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c _6A than to cyanobacterial cytochrome c ₆. In this article, we conclude that the cytochrome c ₆-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c ₆ and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c ₆ and plastocyanin. In any way, the Cyt c ₆-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.     

Effect of nutritional factors on the culture of Nostoc sp. as a source of phycobiliproteins

Summary The effect of nutritional factors in a new culture medium (BW₃) is described for the cyanobacterium Nostoc sp. The growth of Nostoc sp. was higher in BW₃ than in control media currently used for cyanobacteria. With medium BW₃ the content of the pigment c-phycocyanin depended on the culture conditions employed, particularly the nature of the nitrogen and carbon sources. Higher amounts of c-phycocyanin amounting to 20.1% on a dry-weight basis were accumulated when both sources were supplied in the gas phase of the culture. The phycobiliproteins of Nostoc sp. were resolved into two components: c-phycocyanin (_max = 614 nm) and allophycocyanin (_max = 652 nm). The phycobiliprotein composition was 30% allophycocyanin and 70% c-phycocyanin. The culture of Nostoc sp. in BW₃ medium seems promising as a source of biomass for the production of natural dyes.