Qin Song

Metagenome of microorganisms associated with the toxic Cyanobacteria Microcystis aeruginosa analyzed using the 454 sequencing platform

In this study, the 454 pyrosequencing technology was used to analyze the DNA of the Microcystis aeruginosa symbiosis system from cyanobacterial algal blooms in Taihu Lake, China. We generated 183 228 reads with an average length of 248 bp. Running the 454 assembly algorithm over our sequences yielded 22 239 significant contigs. After excluding the M. aeruginosa sequences, we obtained 1 322 assembled contigs longer than 1 000 bp. Taxonomic analysis indicated that four kingdoms were represented in the community: Archaea (n = 9; 0.01%), Bacteria (n = 98 921; 99.6%), Eukaryota (n = 373; 3.7%), and Viruses (n = 18; 0.02%). The bacterial sequences were predominantly Alphaproteobacteria (n = 41 805; 83.3%), Betaproteobacteria (n = 5 254; 10.5%) and Gammaproteobacteria (n = 1 180; 2.4%). Gene annotations and assignment of COG (clusters of orthologous groups) functional categories indicate that a large number of the predicted genes are involved in metabolic, genetic, and environmental information processes. Our results demonstrate the extraordinary diversity of a microbial community in an ectosymbiotic system and further establish the tremendous utility of pyrosequencing.

Pyrolytic characteristics and kinetics of the marine green tide macroalgae, Enteromorpha prolifera

The marine macroalgae Enteromorpha prolifera was one of the main algal genera that occurred in the widespread green tides in Qingdao, China, during the summers of 2007, 2008 and 2010. It is thus a plentiful source of biomass and could be used as a biofuel. In this study, the pyrolytic characteristics and kinetics of E. prolifera were investigated using thermogravimetric analysis (TGA) method. Cornstalk and sawdust were used as comparisons. Pyrolytic characteristics were studied using TG-DTG (thermogravimetry-derivative thermogravimetry) curves. Three stages in the pyrolytic process were determined: dehydration, dramatic weight loss and slow weight loss. E. prolifera was pyrolyzed at a lower initial temperature than the two terrestrial biomass forms. The apparent activation energy values for the three types of biomass were calculated and the mechanism functions were determined using 16 different mechanism functions, frequently used in thermal kinetics analysis. Activation energy values varied with mechanism function and the range of activation energy values for E. prolifera, cornstalk, and sawdust were 25–50 kJ/mol, 60–90 kJ/mol and 120–155 kJ/mol, respectively. This indicates that E. prolifera has low thermal stability for pyrolysis and good combustion characteristics.

Optimization of dilute acid hydrolysis of Enteromorpha

Acid hydrolysis is a simple and direct way to hydrolyze polysaccharides in biomass into fermentable sugars. To produce fermentable sugars effectively and economically for fuel ethanol, we have investigated the hydrolysis of Enteromorpha using acids that are typically used to hydrolyze biomass: H2SO4, HCl, H3PO4 and C4H4O4 (maleic acid). 5%(w/w) Enteromorpha biomass was treated for different times (30, 60, and 90 min) and with different acid concentrations (0.6, 1.0, 1.4, 1.8, and 2.2%, w/w) at 121°C. H2SO4 was the most effective acid in this experiment. We then analyzed the hydrolysis process in H2SO4 in detail using high performance liquid chromatography. At a sulfuric acid concentration of 1.8% and treatment time of 60 min, the yield of ethanol fermentable sugars (glucose and xylose) was high, (230.5 mg/g dry biomass, comprising 175.2 mg/g glucose and 55.3 mg/g xylose), with 48.6% of total reducing sugars being ethanol fermentable. Therefore, Enteromorpha could be a good candidate for production of fuel ethanol. In future work, the effects of temperature and biomass concentration on hydrolysis, and also the fermentation of the hydrolysates to ethanol fuel should be focused on.

A transformation model forLaminaria Japonica (Phaeophyta, Laminariales)

A genetic transformation model for the seaweedLaminaria japonica mainly includes the following aspects:1.The method to introduce foreign genes into the kelp,L. japonicaBiolistic bombardment has been proved to be an effective method to bombard foreign DNA through cell walls into intact cells of both sporophytes and gametophytes. The expression ofcat andlacZ was detected in regenerated sporophytes, which suggests that this method could induce random integration of foreign genes.Promoters to drive gene expression2.The CaMV35S promoter was first used by us to induce the expression of GUS gene in brown algae. But results of further studies suggested that CaMV35S could be a tissue-specific promoter. Our use of SV40 promoter resulted in both transient and stable expression oflacZ andcat in sporophytes or gametophytes. No GUS or LacZ background was found in either sporophytes or gametophytes.The regeneration route of transgenic kelpThe regeneration efficiency of explants is still very low. By using female gametophytes as gene hosts and parthenogenesis as regeneration route, CAT activity and LacZ activity were detected in regenerated sporophytes of parthenogenetic kelp. li]4.|The way to select transgenic kelp1.Results of sensitivity tests showed that kelp was only sensitive to chloramphenicol and hygromycin among many antibiotics. The regenerated sporophytes by parthenogenesis were more sensitive to hygromycin than to chloramphenicol. Resistant kelp was created by transforming female gametophytes with pSV40-CAT and stimulating parthenogenesis followed by selection in medium with lethal concentration of chloramphenicol.Safety consideration of transgenic kelpL. japonica was originally introduced from Japan. In China it is a cultured population. The possibility of its negative impact on natural populations is very low. 2) The vectors and target genes used for transformation should be restricted in order to avoid any negative impacts on human health and environment. 3) Specially devised containers (3.6 L, made of 200 μm membrane) were used to ensure that the kelp cannot escape or be eaten by marine animals. 4) To avoid the release of spores, it is very necessary to harvest the kelp at suitable age before the sporangium forms.

High efficiency induction of callus and regeneration of sporophytes ofLaminaria japonica (Phaeophyta)

Four media (PESI solid, MS liquid, MS solid and ASP-C-I solid medium) were used to induce callus from excised tissues of the kelpLaminaria japonica. Only PESI solid medium and MS solid medium produced calli. Modified MS solid medium supplemented with mannitol (3%,W/V), yeast extract (0.1%, W/V), VB2 (0.5 mg/ml), VB12 (0.5 mg/ml), kinetin (0.108 μg/ml) and NAA (1.860μg/ml) showed much better effect on callus induction than non-modified MS solid medium. After 24 days of induction 75.5% of tissues in PESI solid medium showed callus formation. For modified MS solid medium, after three months of induction 67.3% of tissues dedifferentiated into calli. No callus could be found after five months of induction in either MS liquid or ASP-C-I solid medium. When calli were squashed and cultured in N-P enriched autoclaved seawater, MS liquid medium and ASP12-NTA liquid medium (both modified with kelp extract), differentiation of cells and regeneration of sporophytes were only observed in ASP12-NTA medium supplemented with kelp extract. Gametophyte-like filaments formed first, then eggs were released. It was suggested that sporophyte formation could be a process of parthenogenesis. Sterilization techniques in tissue culture ofL. japonica were also tested in this study.

Effects of ultrasonic treatment on female gametophytes ofLaminaria japonica (Phaeophyta)

This study on the effects of ultrasonic treatment on female gametophytes ofLaminaria japonica showed that:1.Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certain period of time, the average length of filamentous female gametophytes was shortened.2.Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with time of treatment. Ultrasonic treatment had harmful effect on cells.3.Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, output of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regeneration of female gametophytes into sporophytes was effected. Female ganetophytes could not recover after too long period of treatment.

Cloning and sequencing of the allophycocyanin genes fromSpirulina maxima (Cyanophyta)

The genes coding for the α-and β-subunit of allophycocyanin (apcA andapcB) from the cyanophyteSpirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotide sequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The amino acid sequence identities betweenS. maxima andS. platensis are 99.4% for α subunit and 100% for β subunit.

Transposable genetic elements inSpirulina and potential applications for genetic engineering

Transposable elements in cyanobacteria are briefly reviewed. Evidence is presented to show that transposable elements inSpirulina platensis is actually reflected on the phenotype change, i e., helical to straight filaments. Transposition intermediates of DNA were isolated from the extrachromosome and the transposition was related to helical variations inSpirulina. Uses of transposable elements for microalgal recombination are discussed based on the transposition mechanism.

Pyrolytic and kinetic analysis of coastal plant Xanthium sibiricum

The fuel properties of coastal plant Xanthium sibiricum were investigated in thermogravimetrics. The distributed activation energy model was employed in the kinetic analysis and a simplified mathematical model that can predict the thermogravimetry curves was proposed. The results show that the initial decomposition temperature tends to increase with the heating rate. The distributed E values ranged from 169.08 to 177.43 kJ/mol, and the frequency factor values ranged from 6.59×108 to 1.22×1012/s at different conversion rates. Furthermore, the prediction made with the simplified mathematical model perfectly matched the experimental data, and the model was found to be simple and accurate for the prediction of devolatilization curves.

Genetic transformation of marine cyanobacterium Synechococcus sp CC9311 (Cyanophyceae) by electroporation

Synechococcus sp. CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation (CA). A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA. The results show that Synechococcus sp. CC9311 cells were sensitive to four commonly used antibiotics: ampicillin, kanamycin, spectinomycin, and chloramphenicol. An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV, using a kanamycin resistance gene as selectable marker, was constructed by recombinant polymerase chain reaction. The plasmid was then transformed into Synechococcus sp. CC9311 via electroporation. High transformation efficiency was achieved at a field strength of 2 kV/cm. DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin. In addition, the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.