Isolation of a novel strain of Cyanobacterium sp.with good adaptation to extreme alkalinity and high polysaccharide yield

The use of high alkaline medium is a feasible way to provide carbon source and prevent biological contamination for the outdoor cultivation of alkaliphilic microalgae and cyanobacteria.A novel cyanobacterial strain was isolated from the open pond of a marine green alga(Picochlorum sp.SCSIO-45015,Sanya,Hainan)and identified as Cyanobacterium sp.SCSIO-45682.The effects of initial sodium bicarbonate(NaHCO_(3))concentrations on the growth and biochemical composition of Cyanobacterium sp.SCSIO-45682 were investigated.The results demonstrated that Cyanobacterium sp.SCSIO-45682 had good adaptation to 16.8-g/L NaHCO_(3)(the same concentration of NaHCO_(3) used in Zarrouk medium for Spirulina).Moreover,the yields of biomass,polysaccharide,chlorophyll a(chl a),and phycocyanin increased under high NaHCO_(3) concentrations.The maximum final biomass concentration of 2.5 g/L was observed at 8.4-g/L NaHCO_(3),while the highest intracellular total saccharide content of 49.2%of dry weight(DW)and exopolysaccharide(EPS)concentration of 93 mg/L were achieved at the NaHCO_(3) concentration of 16.8 g/L.The crude protein content declined under high NaHCO_(3) concentrations,which provide a possible explanation for the accumulation of polysaccharide.This study shows a good potential of alkaliphilic Cyanobacterium sp.SCSIO-45682 as a polysaccharide feedstock.

Characterization of Novel Alkaliphilic Isolate of <i>Bacillus mannanilyticus</i>, Strain IB-OR17, Displaying Chitinolytic and Antifungal Activities

The novel alkaliphilic strain, designated as Bacillus sp. IB-OR17 and isolated from soda lake sediments, was identified and characterized. Isolated strain demonstrated slight antifungal activity against some plant pathogen fungi that are capable to grow under alkaline conditions. Based on its morphological, physiological and biochemical characteristics as well as on 16S rRNA gene analysis data, Bacillus sp. IB-OR17 were related to alkaliphilic species B. mannanilyticus. Such as type species, Bacillus sp. IB-OR17 produced extracellular β-mannanase but additionally it displayed also chitinolytic activity which is a new property reported for this species. Bacillus sp. IB-OR17 grew in pH range 8.0 - 11.0 with maximal intensity under 9.0 - 10.0 but not showed halophilic properties (growth limit under NaCl concentrations < 5%). Maximal production of chitinase is observed at the same pH interval after 96 h of submerged cultivation of the strain. Bacillus sp. IB-OR17 produced chitinase(s) in presence of colloidal chitin as main carbon source and sodium carbonate (0.25% - 1.0%) demonstrating high enzyme yield under enough low concentrations of the substrate (0.20%). Unlike chitinase, β-mannanase was constitutively produced by Bacillus sp. IB- OR17 in presence of various substrates including crab shell chitin. Probable involvement of the enzymes in antifungal activity of Bacillus sp. IB-OR17 is discussed shortly in terms of further researches and application of this strain.

Ectopic Expression of a Bacterium NhaD-type Na^+/H^+ Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaDwere generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na＋ concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD.overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na＋/H＋ antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.

Comparative proteome analysis of alkaliphilic Bacillus sp.N16-5 grown on different carbon sources

To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.

Isolation, identification and primary application of bacteria from putrid alkaline silica sol

The putrefaction of alkaline silica sol was investigated in this paper. The total colony numbers in three alkaline silica sol samples were 1.47 ×^ 105, 1.25× 10^4, and 9.45 × 10^4 cfu. mL 1, respectively. The salt- and alkalitolerant strains were isolated and selected using nutrient agar medium at 2.5% salinity and pH 9.5. Basic morphological, physiological and biochemical tests were conducted to confirm the preliminary characterizations of the strains. Based on API 50 CH test and 16S rDNA gene sequence analysis, the isolated strains were finally identified as Exiguobacterium aurantiacum, Cyclobacteriaceae bacterium, Microbacterium sp., Acinetobacter sp., Stenotrophomonas maltophilia and Bacillus thuringiensis. The survivability of the strains under different conditions such as salinities, acidities and temperatures was also studied. Some suitable methods for degerming, such as product pipe steam sterilization and regular canister cleaning, were proposed. To explore the possibility of isolates in industrial application, their alkaline protease and amylase production abilities were preliminarily studied. Five strains produced alkaline protease, whereas two strains produced alkaline amylase. Thus, understanding of the putrefaction on alkaline silica sol would be beneficial for improving industrial production.

On the diversity of fungi from soda soils

The diversity of filamentous fungi that can grow at high ambient pH values(i.e.,8–11)remains largely understudied.Here we study 100 alkalitolerant and alkaliphilic isolates from the soils around the basin of soda lakes in Asia and Africa to assess the major evolutionary lineages and morphologies pertinent to the alkaliphilic trait in filamentous fungi.The Emericellopsis lineage(Hypocreales,Hypocreomycetidae),along with Plectosphaerellaceae(Hypocreomycetidae),Pleosporaceae(Dothideomycetes),Chaetomiaceae(Sordariomycetidae)families appeared to be overrepresented with strong alkalitolerants and effective alkaliphiles.In particular,Sodiomyces species(Plectosphaerellaceae),Acrostalagmus luteoalbus(Plectosphaerellaceae),Emericellopsis alkalina(Hypocreales),Thielavia sp.(Chaetomiaceae),and Alternaria sect.Soda(Pleosporaceae)grew best at high ambient pH.The pH tolerance of Chordomyces antarcticum,Acrostalagmus luteoalbus and some other species was largely affected by the presence of extra Na+in the growth medium.Moderate alkalitolerants included Scopulariopsis members(Microascales),Fusarium,Cladosporium,and many asexual acremonium-like species from Bionectriaceae.Weak alkalitolerants were represented by sporadic isolates of Penicillium,Purpureocillium lilacinum,and Alternaria alternata species,with the growth optimum at neutral or acidic pH.Weak alkalitolerants develop loose dry chains of spores easily dispersed by air.Their presence at low frequency with the growth optimum at neutral or acidic pH leads us to treat them as transient species in the alkaline soils,as those are also ubiquitous saprobes in normal soils.Our phylogenetic analyses show that the alkaliphilic trait in filamentous fungi has evolved several times.Several lineages harboring strong alkalitolerants derived from the known marine-borne fungi(Emericellopsis,Alternaria sect.Phragmosporae),or fall within the fungi associated with halophytic grasses(Pleosporaceae).Soda soils contain a diversity of fungi that range from weak alkalitolerant to alkaliphilic,which in few cases is associated with darkly pigmented mycelium and formation of microsclerotia.The alkaliphilic trait is spread throughout the Ascomycota,and usually juxtaposes with slime-covered polyphyletic acremonium-,verticillium-,gliocladium-types of asexual morphology,hyphae aggregating in chords,and enclosed fruit bodies.