Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Roseinatronobacter alkalisoli sp. nov., an alkaliphilic bacterium isolated from soda soil, and genome-based reclassification of the genera Rhodobaca and Roseinatronobacter.

The genera Rhodobaca and Roseinatronobacter are phylogenetically related genera within the family Paracoccaceae. Species of these genera were described using 16S rRNA gene-based phylogeny and phenotypic characteristics. However, the 16S rRNA gene identity and phylogeny reveal the controversy of the taxonomic status of these two genera. In this work, we examined the taxonomic positions of members of both genera using 16S rRNA gene phylogeny, phylogenomic analysis and further validated using overall genome-related indexes, including digital DNA-DNA hybridization, average nucleotide identity, average amino acid identity and percentage of conserved proteins. Based on phylogenetic and phylogenomic results, the current four species of the two genera clustered tightly into one clade with high bootstrap values, suggesting that the genus Rhodobaca should be merged with Roseinatronobacter. In addition, a novel species isolated from a soda soil sample collected from Anda City, PR China, and designated as HJB301T was also described. Phenotypic, chemotaxonomic, genomic and phylogenetic properties suggested that strain HJB301T (=CCTCC AB 2021113T=KCTC 82977T) represents a novel species of the genus Roseinatronobacter, for which the name Roseinatronobacter alkalisoli sp. nov. is proposed.

Influences of lower pH on phytoplankton growth in alkaline lakes after water transfer: Insights from a coupled hydrodynamic-algal ecological model and experimental analysis.

Alkaline lakes with high pH and unique ecological communities often face water-level drawdown and ecological degradation problems due to climatic and hydrologic factors. Water transfer is becoming a popular method for solving these problems. However, a high pH is often considered the key to maintaining the stability of alkaliphilic algal communities, and a lower pH induced by water transfer from a neutral-pH river may threaten ecosystems in alkaline lakes. To explore the response characteristics of phytoplankton in alkaline lakes to pH changes, we conducted cultivation experiments on one species of dominant Cyanobacteria and one species of dominant Chlorophyta from alkaline lakes under different pH conditions. Subsequently, we constructed a coupled hydrodynamic and algal mathematical model considering the effect of pH to predict the dynamic changes in phytoplankton in a typical alkaline lake under water-transfer conditions. Both species are basophilic, and pH has a "low-inhibition and high-promotion" effect on their growth. A lower pH is detrimental to cyanobacterial growth and competitiveness, which may cause Cyanobacteria to lose their dominance in weakly alkaline environments with a pH < 8.5; additionally, water transfer causes a decrease in the total biomass and proportion of Cyanobacteria in Lake Chenghai, with decreases induced by pH changes accounting for 13.4% and 70.1%, respectively. The decrease in pH is the main reason for the decrease in dominance of Cyanobacteria after water transfer. These results provide a basic summary of the effects of pH changes on the algal growth in alkaline lakes and are a useful for formulating ecological water-transfer strategies for alkaline lakes.

Alkalimonas mucilaginosa sp. nov. and Alkalimonas cellulosilytica sp. nov. isolated from alkaline Lonar lake, India.

Two alkaliphilic, Gram-stain-negative bacterial strains (MEB004T and MEB108T) were isolated from water samples collected from Lonar lake, India. The phylogenetic analysis of their 16S rRNA gene sequences showed the highest similarity to A. delamerensis DSM 18314T (98.4%), followed by A. amylolytica DSM 18337T and A. collagenimarina JCM 14267T (97.9%). The genome sizes of strains MEB004T and MEB108T were determined to be 3,858,702 and 4,029,814 bp, respectively, with genomic DNA G + C contents of 51.4 and 51.9%. Average Nucleotide Identity, DNA-DNA Hybridization and Amino Acid Identity values between strains (MEB004T and MEB108T) and A. amylolytica DSM 18337T were (82.3 and 85.5), (25.0 and 29.2) and (86.7 and 90.2%). Both novel strains produced industrially important enzymes, such as amylase, lipase, cellulase, caseinase, and chitinase at pH 10 evidenced by the genomic presence of carbohydrate-active enzymes encoding genes. Genomic analyses further identified pH tolerance genes, affirming their adaptation to alkaline Lonar Lake. Dominant fatty acids were Summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, Summed feature 3, Sum In Feature 2 and C12:0 3OH. The prevalent polar lipids included phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The major respiratory quinone was ubiquinone-8. Based on the polyphasic data, we propose the classification of strains MEB004T and MEB108T as novel species within the genus Alkalimonas assigning the names Alkalimonas mucilaginosa sp. nov. and Alkalimonas cellulosilytica sp. nov., respectively. The type strains are MEB004T (= MCC 5208T = JCM 35954T = NCIMB 15460T) and MEB108T (= MCC 5330T = JCM 35955T = NCIMB 15461T).

Anaerobic microbial metabolism in soil columns affected by highly alkaline pH: Implication for biogeochemistry near construction and demolition waste disposal sites.

Construction and demolition wastes (CDWs) have become a significant environmental concern due to urbanization. CDWs in landfill sites can generate high-pH leachate and various constituents (e.g., acetate and sulfate) following the dissolution of cement material, which may affect subsurface biogeochemical properties. However, the impact of CDW leachate on microbial reactions and community compositions in subsurface environments remains unclear. Therefore, we created columns composed of layers of concrete debris containing-soil (CDS) and underlying CDW-free soil, and fed them artificial groundwater with or without acetate and/or sulfate. In all columns, the initial pH 5.6 of the underlying soil layer rapidly increased to 10.8 (without acetate and sulfate), 10.1 (with sulfate), 10.1 (with acetate), and 8.3 (with acetate and sulfate) within 35 days. Alkaliphilic or alkaline-resistant microbes including Hydrogenophaga, Silanimonas, Algoriphagus, and/or Dethiobacter were dominant throughout the incubation in all columns, and their relative abundance was highest in the column without acetate and sulfate (50.7-86.6%). Fe(III) and sulfate reduction did not occur in the underlying soil layer without acetate. However, in the column with acetate alone, pH was decreased to 9.9 after day 85 and Fe(II) was produced with an increase in the relative abundance of Fe(III)-reducing bacteria up to 9.1%, followed by an increase in the methanogenic archaea Methanosarcina, suggestive of methanogenesis. In the column with both acetate and sulfate, Fe(III) and sulfate reduction occurred along with an increase in both Fe(III)- and sulfate-reducing bacteria (19.1 and 17.7%, respectively), while Methanosarcina appeared later. The results demonstrate that microbial Fe(III)- and sulfate-reduction and acetoclastic methanogenesis can occur even in soils with highly alkaline pH resulting from the dissolution of concrete debris.

The improved purification technique for isolation of the novel CGTase from the alkaliphilc strain Caldalkalibacillus mannanilyticus IB-OR17-B1.

Cyclodextrin-glucanotransferase (CGTase, EC 2.4.1.19) is a multifunctional enzyme that catalyzes many enzymatic reactions including cyclization, binding, disproportionation and hydrolysis reactions, playing an important role in the enzymatic synthesis of compounds that are widely used in agriculture, pharmaceuticals, food, chemical and biotechnology industries. The present research is aimed to optimize the purification protocol for the extracellular CGTase of alkaliphilc bacterial strain Caldalkalibacillus mannanilyticus IB-OR17-B1 guaranteeing the enzyme homogeneity and its high yield. The improved combination of ultrafiltration and corn-starch (5% w/v) affinity sorption techniques resulted to mild and rapid isolation of electrophoritically homogenic enzyme at 18 × increase of its specific activity and yield 56%. The developed two-step procedure instead the practiced tree-step one using commonly ion-exchange chromatography as final purification technique highly contributes in advance of cost-effectiveness for industrial production and isolation of valuable CGTases.

Structural dynamics and mechanistic action guided engineering of lipolytic enzymes.

Lipases have been established as important biocatalysts in several industrial applications, owing to their diverse substrate specificity. The availability of data on three-dimensional crystal structures for various lipases offers an opportunity for modulating their structural and functional aspects to design and engineer better versions of lipases. With the aim of investigating the structural components governing the extremophilic behavior of lipases, structural analysis of microbial lipases was performed using advanced bioinformatics and molecular dynamics simulation approaches. In sequences and functionally distinct alkaliphilic and thermophilic lipases were investigated for their functional properties to understand the distinguishing features of their structures. The alkaliphilic lipase from Bacillus subtilis (LipA) showed conformational changes in the loop region Ala132-Met137, subsequently, the active site residue His156 shows two conformations, toward the active site nucleophilic residues Ser77 and away from the Ser77. Interestingly, the active site of LipA is more solvent-exposed and can be correlated with the adoption of an open conformation which might extend and expose the active site region to solvents during the catalysis process. Furthermore, the MD simulation of thermophilic lipase from marine Streptomyces (MAS1) revealed the role of N- and C-terminal regions with disulfide bridges and identified a metal ion binding site that facilitates the enzyme stability. The novel thermo-alkaliphilic lipase can be designed to integrate the stability features of MAS1 into the alkaliphilic LipA. These structural-level intrinsic characteristics can be used for lipase engineering to amend the lipase activity and stability as per the requirements of the industrial processes.

The Laccase of Myrothecium roridum VKM F-3565: A New Look at Fungal Laccase Tolerance to Neutral and Alkaline Conditions.

Most of the currently known fungal laccases show their maximum activity under acidic environmental conditions. It is known that a decrease in the activity of a typical laccase at neutral or alkaline pH values is the result of an increase in the binding of the hydroxide anion to the T2/T3 copper center, which prevents the transfer of an electron from the T1 Cu to the trinuclear copper center. However, evolutionary pressure has resolved the existing limitations in the catalytic mechanism of laccase, allowing such enzymes to be functionally active under neutral/alkaline pH conditions, thereby giving fungi an advantage for their survival. Combined molecular and biochemical studies, homological modeling, calculation of the electrostatic potential on the Connolly surface at pH 5.0 and 7.0, and structural analysis of the novel alkaliphilic laccase of Myrothecium roridum VKM F-3565 and alkaliphilic and acidophilic fungal laccases with a known structure allowed a new intramolecular channel near the one of the catalytic aspartate residues at T2-copper atom to be found. The amino acid residues of alkaliphilic laccases forming this channel can presumably serve as proton donors for catalytic aspartates under neutral conditions, thus ensuring proper functioning. For the first time for ascomycetous laccases, the production of new trimeric products of phenylpropanoid condensation under neutral conditions has been shown, which could have a potential for use in pharmacology.

Taxogenomics of Alkalihalobacterium chitinilyticum sp. nov.: an alkaliphilic chitin degrading bacterial strain isolated from Lonar Lake, India, with potential biotechnological applications.

A novel chitin degrading alkaliphilic bacterial strain (MEB 203 T) was isolated from sediment collected from Lonar lake, India. The strain exhibited its maximum growth at a temperature of 37 °C, with an optimal pH of 10 and a NaCl concentration of 2%. 16S rRNA gene based phylogenetic tree showed that strain was closely related to Alkalihalobacterium elongatum MCC 2982 T (98.64% similarity) followed by A. alkalinitrilicum DSM 22532 T (97.84% similarity). The genome size was 4.9 Mb with DNA G + C content of 37.7%. The dDDH value between strain MEB 203 T and A. elongatum MCC 2982 T was 26.4 ± 2.4% while OrthoANI value was 82.1%. Genome analysis revealed the presence of genes responsible for L-ectoine and cation/proton antiporter which may facilitate growth of strain in alkaline-saline habitat of Lonar lake. Strain MEB 203 T was able to utilize complex sugars such as chitin, cellulose, and starch as a carbon source at alkaline conditions which was also corroborated from the genomic presence of carbohydrate active enzymes (CAZymes). It was also able to produce biotechnologically important enzymes such as lipases and proteases which were stable at pH (9-10). The bacterium is majorly composed of C15:0 iso, C16:0 iso, and C17:0 iso (> 10%) fatty acids while diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipid (PL3) were identified as the predominant polar lipids. Based on differential physiological, biochemical, and genomic features of strain MEB 203 T, a novel species Alkalihalobacterium chitinilyticum sp. nov. (Type strain MEB 203 T = MCC 3920 T = NCIMB 15407 T = JCM 35078 T) is proposed.

Genomic insights of an alkaliphilic bacterium Halalkalibacter alkaliphilus sp. nov. isolated from an Indian Soda Lake.

An alkaliphilic, Gram-stain-positive, non-motile, rod-shaped, and spore forming bacterial strain (MEB205T) was isolated from sediment sample collected from Lonar lake, India. The strain grew optimally at pH 10, NaCl concentration of 3.0% at 37 °C. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain MEB205T belonged to the genus Halalkalibacter in the family Bacillaceae and shared the highest sequence similarity with H. okhensis Kh10-101T (98.9%) followed by H. wakoensis N-1 T (98.7%). The assembled genome of strain MEB205T has a total length of 4.8 Mb with a G + C content of 37.8%. The dDDH and OrthoANI values between strain MEB205T and H. okhensis Kh10-101 T were 29.1% and 84.3%, respectively. Furthermore, the genome analysis revealed the presence of antiporter genes (nhaA and nhaD) and L-ectoine biosynthesis gene required for survival of the strain MEB205T in alkaline-saline habitat. The major fatty acid was C15:0 anteiso, C16:0 and C15:0 iso (> 10.0%). Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. meso-diaminopimelic acid was diagnostic diamino acid for cell wall peptidoglycan. Based on the polyphasic taxonomic studies, strain MEB205T represent a novel species of the genus Halalkalibacter for which the name Halalkalibacter alkaliphilus sp. nov. (Type strain MEB205T = MCC 3863 T = JCM 34004 T = NCIMB 15406 T) is proposed.

Integrating bioelectrochemical system with aerobic bioreactor for organics removal and caustic recovery from alkaline saline wastewater.

Bioelectrochemical systems (BES) are increasingly being explored as an auxiliary unit process to enhance conventional waste treatment processes. This study proposed and validated the application of a dual-chamber bioelectrochemical cell as an add-on unit for an aerobic bioreactor to facilitate reagent-free pH-correction, organics removal and caustic recovery from an alkaline and saline wastewater. The process was continuously fed (hydraulic retention time (HRT) of 6 h) with a saline (25 g NaCl/L) and alkaline (pH 13) influent containing oxalate (25 mM) and acetate (25 mM) as the target organic impurities present in alumina refinery wastewater. Results suggested that the BES concurrently removed the majority of the influent organics and reduced the pH to a suitable range (9-9.5) for the aerobic bioreactor to further remove the residual organics. Compared to the aerobic bioreactor, the BES enabled a faster removal of oxalate (242 ± 27 vs. 100 ± 9.5 mg/L.h), whereas similar removal rates (93 ± 16 vs. 114 ± 23 mg/L.h, respectively) were recorded for acetate. Increasing catholyte HRT from 6 to 24 h increased the caustic strength from 0.22% to 0.86%. The BES enabled caustic production at an electrical energy demand of 0.47 kWh/kg-caustic, which is a fraction (22%) of the electrical energy requirement for caustic production using conventional chlor-alkali processes. The proposed application of BES holds promise to improve environmental sustainability of industries in managing organic impurities in alkaline and saline waste streams.

Halomonas salinarum sp. nov., a moderately halophilic bacterium isolated from saline soil in Yingkou, China.

Strain G5-11T, a Gram-negative, moderately halotolerant, facultatively aerobic, motile bacterium was isolated from saline soil collected from Yingkou, Liaoning, China. The cells of strain G5-11T grew in the presence of 3-15% (w/v) NaCl (optimum 5%), at between 4 and 35 °C (optimum 30 °C), and at a pH of 6.0-9.0 (optimum 8.0). The major respiratory quinone was Q-9 and the dominant cellular fatty acids were summed feature 8 (C18:1ω7c/C18:1ω6c), C16:0, and summed feature 3 (C16:1ω7c/C16:1ω6c). The major components of the polar lipid profile were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and unidentified aminolipid. The G + C content of the strain G5-11T genome was 61.0 mol%. The isolated strain G5-11T showed the highest 16S rRNA gene similarity to Halomonas niordiana LMG 31227T and Halomonas taeanensis DSM 16463T, both reaching 98.3%, followed by Halomonas pacifica NBRC 102220T. The results from phenotypic, chemotaxonomic, and phylogenetic analyses showed that strain G5-11T represented a novel species of the genus Halomonas, for which the name Halomonas salinarum sp. nov. was proposed. The type strain of Halomonas salinarum is G5-11T (= CGMCC 1.12051T = LMG 31677T).

Fundicoccus fermenti sp. nov., an indigo-reducing facultative anaerobic alkaliphile isolated from indigo fermentation liquor used for dyeing.

Two facultative anaerobic and facultative alkaliphilic indigo-reducing strains, designated F-1T and F-2, were isolated from indigo fermentation liquor produced from couched woad fermentation-based Indian indigo fermentation fluid. The 16S rRNA gene phylogeny showed that Fundicoccus ignavus WS4937T (99.5%) was the closest neighbour of F-1T. The isolated bacterial cells were Gram-stain-positive and facultative anaerobic coccoids. Strain F-1T grew at between 5 and 37 °C with optimum growth between 28‒32 °C. The isolate grew in a pH range of 7.0‒10.5, with optimum growth between pH 9.0‒10.5. The DNA G+C content was 37.6 mol% (HPLC). The whole-cell fatty acid profile mainly consisted (>10 %) of C16 : 0, C16 : 1 ω9c, C18 : 0 and C18 : 1 ω9c. The digital DNA-DNA hybridization value between strain F-1T and F. ignavus WS4937T was 52.9 %. Based on their physiological and biochemical characteristics, and phylogenetic and genomic data, the isolates can be discriminated from F. ignavus WS4937T. The name Fundicoccus fermenti sp. nov. is proposed. The type strain of this species is F-1T (JCM 34140T=NCIMB 15255T).

Identification and Characterization of a New Cold-Adapted and Alkaline Alginate Lyase TsAly7A from Thalassomonas sp. LD5 Produces Alginate Oligosaccharides with High Degree of Polymerization.

Alginate oligosaccharides (AOS) and their derivatives become popular due to their favorable biological activity, and the key to producing functional AOS is to find efficient alginate lyases. This study showed one alginate lyase TsAly7A found in Thalassomonas sp. LD5, which was predicted to have excellent industrial properties. Bioinformatics analysis and enzymatic properties of recombinant TsAly7A (rTsAly7A) were investigated. TsAly7A belonged to the fifth subfamily of polysaccharide lyase family 7 (PL7). The optimal temperature and pH of rTsAly7A was 30 °C and 9.1 in Glycine-NaOH buffer, respectively. The pH stability of rTsAly7A under alkaline conditions was pretty good and it can remain at above 90% of the initial activity at pH 8.9 in Glycine-NaOH buffer for 12 h. In the presence of 100 mM NaCl, rTsAly7A showed the highest activity, while in the absence of NaCl, 50% of the highest activity was observed. The rTsAly7A was an endo-type alginate lyase, and its end-products of alginate degradation were unsaturated oligosaccharides (degree of polymerization 2-6). Collectively, the rTsAly7A may be a good industrial production tool for producing AOS with high degree of polymerization.

Evaluating the potential of thermo-alkaliphilic microbial consortia for azo dye biodegradation under anaerobic-aerobic conditions: Optimization and microbial diversity analysis.

Wastewaters in textile industry are mainly characterized by higher pH, color, salt and chemical oxygen demand (COD) values, which are environmentally undesirable. Among these textile effluent characteristics, color removal is the most challenging task. In this study, the potential of Rift Valley halotolerant and thermo-alkaliphilic microbial consortia (collected from Shala hot spring located in Ethiopia) for azo dye biodegradation under anaerobic-aerobic conditions were evaluated. Optimization and microbial diversity analysis were done using Reactive Red 141. Under optimum conditions of pH (9), temperature (55 °C), salinity (0.5%), and nutrients, microbial consortia can remove >98% color and 92.7 ± 7.3% COD under anaerobic and aerobic conditions, respectively. In addition, the consortia was capable of decolorizing initial dye concentrations of 100-1000 mg/L, and various dye types including Everzol Blue LX, RY 84, RR 239, RB 198 and RY 700. The 16S rRNA gene sequence results showed that Bacteroidetes (25.3%) > Proteobacteria (21.0%) > Chloroflexi (18.5%) > Halobacterota (6.2%) dominant phyla. Based on the findings, non-color effluent adapted Rift Valley halotolerant and thermo-alkaliphilic bacterial consortia can be a potential candidate for bioremediation of textile and other industries characterized by higher salinity, temperature and pH.

Exploring Peptaibol's Profile, Antifungal, and Antitumor Activity of Emericellipsin A of Emericellopsis Species from Soda and Saline Soils.

Features of the biochemical adaptations of alkaliphilic fungi to exist in extreme environments could promote the production of active antibiotic compounds with the potential to control microorganisms, causing infections associated with health care. Thirty-eight alkaliphilic and alkalitolerant Emericellopsis strains (E. alkalina, E. cf. maritima, E. cf. terricola, Emericellopsis sp.) isolated from different saline soda soils and belonging to marine, terrestrial, and soda soil ecological clades were investigated for emericellipsin A (EmiA) biosynthesis, an antifungal peptaibol previously described for Emericellopsis alkalina. The analysis of the Emericellopsis sp. strains belonging to marine and terrestrial clades from chloride soils revealed another novel form with a mass of 1032.7 Da, defined by MALDI-TOF Ms/Ms spectrometers, as the EmiA lacked a hydroxyl (dEmiA). EmiA displayed strong inhibitory effects on cell proliferation and viability of HCT 116 cells in a dose- and time-dependent manners and induced apoptosis.

Bacillus alkalicellulosilyticus sp. nov., isolated from extremely alkaline bauxite residue (red mud) site.

A Gram-stain-negative, rod-shaped, facultatively anaerobic, motile and spore-forming strain designated FJAT-44921T was isolated from red mud collected from Chiping County, Shandong Province, China. The 16S rRNA gene sequence result showed that strain FJAT-44921T shared a low sequence identity (96.6%) with the members of the genus Bacillus. Growth was observed at pH 8.0-10.0 (optimum pH 9.0), 10-40 °C (optimum 20-25 °C) with 0-8% (v/w %) NaCl (optimum 4-6 v/w %). FJAT-44921T consists of MK-7 as the isoprenoid quinone and meso-2,6-diaminopimelic acid as the cell-wall diamino acid. The predominant fatty acids were anteiso-C15:0, iso-C15:0, C16:0, and anteiso-C17:0. The polar lipids were diphosphatidylglycerol, phosphatidyl glycerol, phosphatidylmethylethanolamine, unidentified phospholipid, and unidentified aminophospholipid. The genomic DNA G + C content was 37.3 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between FJAT-44921T and other closely related Bacillus members were lower than the recognized threshold values of ANI (95-96%) and dDDH (70%) recommended as the criterion for interspecies identity. The type strain is FJAT-44921T (=CCTCC AB 2016196T =DSM 104630T).

Alkalicella caledoniensis gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium isolated from 'La Crouen' alkaline thermal spring, New Caledonia.

A novel anaerobic, alkaliphilic, mesophilic, Gram-stain-positive, endospore-forming bacterium was isolated from an alkaline thermal spring (42 °C, pH 9.0) in New Caledonia. This bacterium, designated strain LB2T, grew at 25-50 °C (optimum, 37 °C) and pH 8.2-10.8 (optimum, pH 9.5). Added NaCl was not required for growth (optimum, 0-1 %) but was tolerated up to 7 %. Strain LB2T utilized a limited range of substrates, such as peptone, pyruvate, yeast extract and xylose. End products detected from pyruvate fermentation were acetate and formate. Both ferric citrate and thiosulfate were used as electron acceptors. Elemental sulphur, nitrate, nitrite, fumarate, sulphate, sulfite and DMSO were not used as terminal electron acceptors. The two major cellular fatty acids were iso-C15 : 0 and C16 : 0. The genome consists of a circular chromosome (3.7 Mb) containing 3626 predicted protein-encoding genes with a G+C content of 36.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate is a member of the family Proteinivoraceae, order Clostridiales within the phylum Firmicutes. Strain LB2T was most closely related to the thermophilic Anaerobranca gottschalkii LBS3T (93.2 % 16S rRNA gene sequence identity). Genome-based analysis of average nucleotide identity and digital DNA-DNA hybridization of strain LB2T with A. gottschalkii LBS3T showed respective values of 70.8 and 13.4 %. Based on phylogenetic, genomic, chemotaxonomic and physiological properties, strain LB2T is proposed to represent the first species of a novel genus, for which the name Alkalicella caledoniensis gen. nov., sp. nov. is proposed (type strain LB2T=DSM 100588T=JCM 30958T).

Paraliobacillus salinarum sp. nov., isolated from saline soil in Yingkou, China.

A novel Gram-stain-positive, facultatively aerobic, slightly halophilic, endospore-forming bacterium, designated G6-18T, was isolated from saline soil collected in Yingkou, Liaoning, PR China. Cells of strain G6-18T grew at 10-37 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 8.0) and in the presence of 2-15 % (w/v) NaCl (optimum, 5 %). The strain could be clearly distinguished from the related species of the genus Paraliobacillus by its phylogenetic position and biochemical characteristics. It presented MK-7 as the major quinone and the dominant cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0, C16 : 0 and iso-C14 : 0. The polar lipids consisted of diphosphatidylglycerol and phosphatidylglycerol as the major components. The G+C content of strain G6-18T genome was 35.3 mol%. 16S rRNA analysis showed that strain G6-18T had the highest similarity to Paraliobacillus ryukyuensis DSM 15140T, reaching 97.0 %, followed by Paraliobacillus quinghaiensis CGMCC 1.6333T with a value of 96.3 %. The average nucleotide identity values between strain G6-18T and Paraliobacillus ryukyuensis DSM 15140T, Paraliobacillus sedimins KCTC 33762T, Paraliobacillus quinghaiensis CGMCC 1.6333T and Paraliobacillus zengyii DSM 107811T were 74.3, 72.0, 73.2 and 72.8 %, respectively, and the digital DNA-DNA hybridization values between strain G6-18T and the neighbouring strains were 15.6, 13.8, 14.2 and 14.2 %, respectively. Based on phenotypic, chemotaxonomic and phylogenetic inferences, strain G6-18T represents a novel species of the genus Paraliobacillus, for which the name Paraliobacillus salinarum sp. nov. (=CGMCC 1.12058T=DSM 25428T) is proposed.

Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria.

OBJECTIVES: To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering RESULTS: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-ß1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose. CONCLUSIONS: A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.