Type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus

The hair‐like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo‐proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm‐promoting function in type IV pili‐producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non‐piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well‐studied type IV pili‐producing heterotrophic bacteria.     

Isolation and Characterization of Taiwanese Heterotrophic Microalgae: Screening of Strains for Docosahexaenoic Acid (DHA) Production

Marine heterotrophic microalgal species which are potentially rich in docosahexaenoic acid (DHA, C22:6n−3) have been found in Taiwan; however, there was a lack of detailed analysis and characterization of these indigenous algae which is needed for the development of commercial applications. Hence, the objective of this study was to screen DHA-rich heterotrophic microalgae species indigenous to Taiwan for commercial purposes. Heterotrophic microalgae from a variety of marine habitats were isolated, cultivated, and then identified according to their 18S rRNA gene sequences and morphological characteristics. A comparison was made of their fatty acid profiles, fatty acid content, and amount of biomass. For the strain with highest DHA yield, the optimal growth conditions were determined in order to establish the best fermentation conditions for scale-up. In this study, 25 heterotrophic microalgal strains were successfully isolated from marine habitats around Taiwan. All of the isolated strains showed a close phylogenic relationship with the Thraustochytriaceae family according to their 18S rRNA gene sequences. GC/MS analysis discerned seven distinctive fatty acid profiles of these strains, with the production of eicosapentaenoic acid (C20:5n−3) ranging from 0.02 to 2.61 mg L⁻¹, and DHA ranging from 0.8 to 18.0 mg L⁻¹. An Aurantiochytrium strain BL10 with high DHA production was subsequently chosen for further manipulation. Under optimal growth conditions it could produce up to 59.0 g of dry biomass per liter of culture, with dry biomass containing 73% total fatty acid and 29% DHA, revealing BL10 as an excellent source of microbial DHA.     

Biological dinitrogen fixation by selected soil cyanobacteria as affected by strain origin,morphotype, and light conditions

The potential for N₂ fixation by heterocystous cyanobacteria isolated from soils of different geographical areas was determined as nitrogenase activity (NA) using the acetylene reduction assay. Morphology of cyanobacteria had the largest influence on NA determined under light conditions. NA was generally higher in species lacking thick slime sheaths. The highest value (1446 nmol/h C₂H₄ per g fresh biomass) was found in the strain of branched cyanobacterium Hassalia (A Has1) from the polar region. A quadratic relationship between NA and biomass was detected in the Tolypothrix group under light conditions. The decline of NA in dark relative to light conditions ranged from 37 to 100 % and differed among strains from distinct geographical areas. Unlike the NA of temperate and tropical strains, whose decline in dark relative to light was 24 and 17 %, respectively, the NA of polar strains declined to 1 % in the dark. This difference was explained by adaptation to different light conditions in temperate, tropical, and polar habitats. NA was not related to the frequency of heterocysts in strains of the colony-forming cyanobacterium Nostoc. Colony morphology and life cycle are therefore more important for NA then heterocyst frequency. NA values probably reflect the environmental conditions where the cyanobacterium was isolated and the physiological and morphological state of the strain.     

Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions

Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs'' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

The production of biomass and phycobiliprotein pigments in filamentous cyanobacteria: the impact of light and carbon sources

Cyanobacteria are recognized as producers of bioactive substances and phycobiliproteins, whose medicinal and functional food properties have led to increased interest in recent years. In the present study, the biomass production and phycobiliprotein content in cyanobacterial strains belonging to Anabaena, Nostoc and Spirulina genera were investigated under the conditions of continuous illumination and mixotrophic nutrition. The results showed that biomass production was strongly stimulated by continuous light in Spirulina strains (4.5-fold), and by organic carbon sources in N₂-fixing strains (2.1–2.8-fold). The strategy of cells to accumulate primarily blue pigment phycocyanin and bluish green allophycocyanin was revealed under tested conditions. Furthermore, in the case of Spirulina S1 grown with glycerol, the culture medium became dense and changed its colour to pink, which may indicate the release of compounds including pigment(s) outside the cell, the phenomenon that seem to be rare among cyanobacteria. Moreover, under continuous light, in this strain the highest biomass level of 4.0 mg/mL was achieved, wherein phycocyanin and allophycocyanin content was increased 12- and 16-fold, respectively, which indicates the high potential of this strain for further investigation.     

Novel eubacteria able to grow on carbon disulfide

Four eubacterial strains able to grow on carbon disulfide (CS₂) as sole energy substrate were isolated from soil and leaves of the CS₂-producing tree Quercus lobata. Three of the isolates (strains KS1, KS2, and KL1) were gram-negative, facultatively methylotrophic, and heterotrophic, and capable of growth on a wide range of inorganic and organic sulfur compounds. Biochemical and physiological properties differed slightly among the three strains, but all are proposed to be novel thiobacillus species. Growth yields on CS₂ in batch and chemostat culture ranged from 3.3 g dry wt/mol CS₂ (batch) to a maximum growth yield (Y_max) of 11.1 g dry wt/mol (chemostat). Chemostat data for two of the strains growing, autotrophically on thiosulfate gave Y_max values of 7.4 and 7.1 g dry wt/mol, which fall within the range observed with thiobacilli. The three new Thiobacillus strains had DNA containing 39.8 (KS2), 47.8 (KS1), and 50.5 (KL1) mol% G+C. All three were unusual in being able to grow not only on thiosulfate (aerobically or with denitrification), but also on CS₂, carbonyl sulfide and methylated sulfides as sole energy substrates, and one was unique in being able to grow also on substituted thiophenes. They are the first organisms described to be capable, of anaerobic growth with denitrification on CS₂. The fourth isolate (strain KL2) was gram-positive non-motile and nonspore-forming, with 39.0 mol% G+C. It had a restricted range of sulfur-containing growth substrates, could not grow methylotrophically or on autotrophic substrates other than CS₂, and is not yet classifiable These organisms extend the range of eubacteria known to be capable of CS₂ breakdown and demonstrate that several types of facultatively chemolithotrophic bacteria, able to grow exclusively on CS₂, are associated with a CS₂-producing plant.     

Innovative development of membrane sparger for carbon dioxide supply in microalgae cultures

The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO₂) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO₂ supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L⁻¹ d⁻¹). The highest values of biomass concentration (1.98 g L⁻¹), biomass productivity (131.8 mg L⁻¹ d⁻¹), carbon in biomass (47.9% w w⁻¹), CO₂ fixation rate (231.6 mg L⁻¹ d⁻¹), and CO₂ use efficiency (80.5% w w⁻¹) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO₂ supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w⁻¹). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.     

A <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain HPC248 from an effluent treatment plant with antimicrobial activity

This study reports the identification and demonstration of an organism with antimicrobial activity isolated from activated biomass of an effluent treatment plant (ETP) treating wastewater containing pesticides. While assessing the heterotrophic diversity of biomass collected from ETP, clear zones were observed on Luria Bertani plates. The bacterial isolate producing the zone as well as the bacterial cells surrounding the zone were isolated and purified by sub-culturing. Both isolates were identified by partial sequencing of the 16S rDNA clone. Presence of antimicrobial activity was demonstrated against various laboratory strains, isolated from different treatment plants and also against waterborne pathogens. The isolate that produced antimicrobial activity was identified as Bacillus subtilis strain HPC248 and the sensitive strain was identified as Bacillus sphaericus strain HPC249.     

Biogenic synthesis of gold nanoparticle using fractioned cellular components from eukaryotic algae and cyanobacteria

In the present investigation fractioned cellular components like intact pigment bearing thylakoids/chloroplasts, carotenoids, protein, polysaccharides were extracted from the cyanobacterium Anabaena sphaerica and green alga Chlorococcum infusionum. Each of these extracts was used separately in search for efficient reducing agents during gold nanoparticle (GNP) production in pro‐ and eukaryotic algal cell systems. The whole biomass and extracted compounds or cellular structures were exposed in 25 mg L^?1 aqueous hydrogen tetrachloroaurate solutions separately at room temperature. Isolated viable chloroplasts from C. infusionum and thylakoids from A. sphaerica were found to be able to reduce gold ions. The protein extracts of both strains were also able to synthesize GNP at 4°C. Extracted polysaccharides of the two strains responded differently. Polysaccharides from A. sphaerica showed positive response in GNP synthesis, whereas no change was observed for C. infusionum. The carotenoids extracts from both strains acted like an efficient reducing agent. Initially the reducing efficiency of these extracted components was confirmed by the appearance of purple color in biomass or in experimental media. The GNPs, synthesized within the biomass were extracted by sonication with sodium citrate. The UV–vis spectroscopy of extracted purple colored suspensions and media showed the absorption bands at approximately 530–540 nm indicating a strong positive signal of GNP synthesis. Transmission electro n microscopy determined the size and shapes of the particles. The X‐ray diffraction study of the synthesized GNP revealed that the 2θ values appeared at 38.2°, 44.5°, 64.8° and 77.8°. Amongst all, isolated thylakoids and chloroplast showed only spherical GNP production with variable size range at pH 4. Monodisperse GNPs were also synthesized by isolated thylakoids and chloroplast at pH 9. A detailed morphological change of gold treated biomass was revealed employing scanning electron microscopy. The fluorescent property of gold loaded cells was studied by fluorescence microscopy.     

A closed solar photobioreactor for cultivation of microalgae under supra-high irradiance: basic design and performance

An account is given of the setting up and use of a novel type of closed tubular photobioreactor at the Academic and University Centre in Nove Hrady, Czech Republic. This "penthouse-roof" photobioreactor was based on solar concentrators (linear Fresnel lenses) mounted in a climate-controlled greenhouse on top of the laboratory complex combining features of indoor and outdoor cultivation units. The dual-purpose system was designed for algal biomass production in temperate climate zone under well-controlled cultivation conditions and with surplus solar energy being used for heating service water. The system was used to study the strategy of microalgal acclimation to supra-high solar irradiance, with values as much as 3.5 times the ambient value, making the approach unique. The cultivation system proved to be fully functional with sufficient mixing and cooling, efficient oxygen stripping and light tracking. Experimental results (measurement of the maximum photochemical yield of PSII and non-photochemical quenching) showed that the cyanobacterium Spirulina (= Arthrospira) platensis cultivated under sufficient turbulence and biomass density was able to acclimate to irradiance values as high as 7 mmol photon m^–2 s^–1. The optimal biomass concentration of Spirulina cultures in September ranged between 1.2 to 2.2 g L^–1, which resulted in a net productivity of about 0.5 g L^–1 d^–1 corresponding to a biomass yield of 32.5 g m^–2 d^–1 (based on the minimum illuminated surface area of the photobioreactor).     

Disturbance,starvation, and overfeeding stresses detected by microbial lipid biomarkers in high-solids high-yield methanogenic reactors

Summary Microbial biomass and community structure of methanogenic anaerobic biomass reactors can be quantitatively monitored by signature, lipid analysis. The eubacterial and eukaryotic polar lipid fatty acids and the methanogen polar lipid ethers are reliable measures of their respective biomasses. The pattern of polar lipid fatty acids yields information on the community structure and metabolic state of the eubacteria and eukaryotes. These biomarker methods were applied over a 2-day feeding cycle of a highly productive batch-fed high-solids anaerobic biomass reactor. It was sampled before feeding, 6 h after feeding (disturbed)., at maximum gas production (healthy, 24 h), and after feedstock utilization (starved, 48h). Relative to the healthy condition, the disturbance of feeding significantly decreased eubacterial biomass and the proportion of unsaturated fatty acids, and increased branched fatty acids and the eubacterial stress biomarker,trans/cis 16: 17. The starved condition was not significantly different from the healthy in biomass or proportions of fatty acids, but did show a significant increase in the proportion of the eubacterial stress biomarkertrans/cis 18: 17. This reactor was compared to a second of the same design which had been overfed and showed significantly less productivity. The overfed reactor had a significantly lower methanogenic biomass,iso-branched fatty acids, and higher eubacterial stress markers Cy17:0 andtrans/cis 18: 17 than the highly productive reactor.     

Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

Alcohol dehydrogenase AdhA plays a role in ethanol tolerance in model cyanobacterium Synechocystis sp. PCC 6803

The protein AdhA from the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) has been previously reported to show alcohol dehydrogenase activity towards ethanol and both NAD and NADP. This protein is currently being used in genetically modified strains of Synechocystis capable of synthesizing ethanol showing the highest ethanol productivities. In the present work, mutant strains of Synechocystis lacking AdhA have been constructed and tested for tolerance to ethanol. The lack of AdhA in the wild-type strain reduces survival to externally added ethanol at lethal concentration of 4% (v/v). On the other hand, the lack of AdhA in an ethanologenic strain diminishes tolerance of cells to internally produced ethanol. It is also shown that light-activated heterotrophic growth (LAHG) of the wild-type strain is impaired in the mutant strain lacking AdhA (∆adhA strain). Photoautotrophic, mixotrophic, and photoheterotrophic growth are not affected in the mutant strain. Based on phenotypic characterization of ∆adhA mutants, the possible physiological function of AdhA in Synechocystis is discussed.

     

Influence of carbon-dioxide on the growth of Spirulina sp. (MCRC-A0003) isolated from Muttukadu backwaters,South India

Growth of Spirulina sp. (MCRC-A0003), a cyanobacterium, was evaluated under different concentrations of carbon-dioxide (CO₂) (4–50 %) in a closed glass photobioreactor. Although significant CO₂ utilization by the cyanobacterial strain was observed up to 50 % concentration, complete utilization was observed only at 4, 10 and 20 % concentrations on 3rd, 6th and 8th day respectively. However, considerable reduction was witnessed in reactors containing 30–50 % CO₂ only between 6th and 9th day. A corresponding increase in the biomass and primary metabolites like chlorophyll-a, carbohydrate and protein were observed. Biomass productivity of Spirulina in reactors sparged with 4, 10 and 20 % CO₂ were 13.7, 43 and 44 % more than that in control reactor without CO₂. While CO₂ increased the levels of primary metabolites in the cyanobacterial cells, it was quite prominent in 10 % CO₂ concentration with the chlorophyll-a, carbohydrate and protein contents were 64, 183 and 626 mg g^?1 respectively. While 10 and 6.6 % increase were noticed in chlorophyll-a and protein, 17 % increase in carbohydrate levels was observed in Spirulina cells, which could be attributed to the conversion of CO₂ to carbohydrate by the cyanobacterium.     

Characterization of bacterial communities associated with Brassica napus L. growing on a Zn-contaminated soil and their effects on root growth

The interaction between plant growth-promoting bacteria (PGPB) and plants can enhance biomass production and metal tolerance of the host plants. This work aimed at isolating and characterizing the cultivable bacterial community associated with Brassica napus growing on a Zn-contaminated site, for selecting cultivable PGPB that might enhance biomass production and metal tolerance of energy crops. The effects of some of these bacterial strains on root growth of B. napus exposed to increasing Zn and Cd concentrations were assessed. A total of 426 morphologically different bacterial strains were isolated from the soil, the rhizosphere, and the roots and stems of B. napus. The diversity of the isolated bacterial populations was similar in rhizosphere and roots, but lower in soil and stem compartments. Burkoholderia, Alcaligenes, Agrococcus, Polaromonas, Stenotrophomonas, Serratia, Microbacterium, and Caulobacter were found as root endophytes exclusively. The inoculation of seeds with Pseudomonas sp. strains 228 and 256, and Serratia sp. strain 246 facilitated the root development of B. napus at 1,000 µM Zn. Arthrobacter sp. strain 222, Serratia sp. strain 246, and Pseudomonas sp. 228 and 262 increased the root length at 300 µM Cd.     

Phylogenetic relatedness among Spirulina and related cyanobacterial genera

Molecular polymorphisms in a selected set of Spirulina and related genera using random primers based on repetitive sequences along with biochemical parameters, led to the unambiguous differentiation of the strains and understanding of their phylogenetic relationships. A combination of 10 sets of dual primers generated 100% distinct polymorphic bands ranging from 150 to 5,000 bp. Total number of fragments ranged from 68 to 159 whereas polymorphic bands ranged from 13 to 32 for different Random Amplified Polymorphic DNA (RAPD) reactions. Spirulina platensis strains, Sp-2 and Sp-3, possessed quite comparable chlorophyll and protein content besides having maximum similarity coefficient (0.88) between them on the basis of RAPD reactions, thus proved to be closely related. Sp-8 (Spirulina from Loktak Lake) having the highest protein content and protein: chlorophyll ratio, showed close similarity with the mutant of Spirulina platensis (Sp-7) on the basis of RAPD analysis. Duncan’s Multiple Range Test (DMRT) ranking for the biochemical parameters were quite closer for the strains of Spirulina and Arthrospira. This is also supported by the cluster analysis based on RAPD data, as the strains of Spirulina and Arthrospira are placed together in the same subcluster in the dendrogram. The comparative closeness among the strains of Lyngbya, Oscillatoria and Phormidium is reflected by the low content of protein and protein: chlorophyll ratio, which is also supported by the dendrogram based upon RAPD; thus, exhibiting the usefulness of multiplex RAPD along with biochemical parameters for the phylogenetic analysis of Spirulina and related genera.     

Evaluation of growth yield of Spirulina (Arthrospira) sp. in photoautotrophic, heterotrophic and mixotrophic cultures

In microbial cultures, both cellular growth rate and yield (defined as the degree of substrate conversion into the biomass) are important. Although effect of culture conditions on growth kinetics has been well documented for various microbial strains, there is almost no literature concerning the effect of environmental conditions on growth equilibrium, expressed as biomass yield coefficients from substrate. The present paper discusses the effect of culture conditions: irradiance (physical substrate) and glucose concentration (chemical substrate) on biomass yield coefficients from two chemical substrates: glucose and nitrate-nitrogen in photoautotrophic, heterotrophic and mixotrophic culture of blue-green alga Spirulina (Arthrospira) sp. The efficiency of substrates incorporation into the biomass can be precisely determined only if the elemental composition of the biomass is known. The experimental results showed that culture conditions had a substantial influence on biomass yield coefficients (biomass yield from glucose and nitrate-nitrogen). It was found that, the increase of irradiance favoured increase of biomass yield coefficient from both, glucose and nitrate-nitrogen. However, in the case of yield from nitrogen in mixotrophic culture, the effect was opposite. The effect of glucose concentration was different: the higher the initial glucose concentration, the lower the biomass yield coefficients from chemical substrates.