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Sulphur reducing bacteria (SRB) and sulphur-oxidizing bacteria (SOB) are predominately involved in reduction and oxidation of sulphates and hydrogen sulphide, respectively, in pond and assist in maintaining healthy pond environment for shrimp culture. Hence, the present study aims to find out SOB two different Penaeus vannamei culture systems. Total 43 isolates from earthen ponds and 38 from plastic-lined ponds were obtained using thiosulphate agar and Starkey medium. The isolates were tested for their efficacy to reduce the pH of the growth media from 8.0 to ≤5.0 within 15 days of incubation. Data revealed the sulphate production was in the range of 2.5 to 195.36 mg/100 ml. The bacterial isolates from earthen pond showed significantly higher (P< 0.05) sulphate ion production compared to bacteria isolated from the plastic-lined pond. Among all these isolated bacteria, fifteen isolates possess autotrophic metabolism. SOBs were able to tolerate salt up to 13%. Morphological and biochemical characterization of isolated SOB revealed that as Bacillus species and Micrococcus sp. were present in the plastic-lined pond isolates whereas, Bacillus sp., Geobacillus sp., Amphibacillus sp., Anoxibacillus sp. and Alkalibacillus sp. were present in the earthen ponds. Thus, the earthen pond has more diverse SOB than in the plastic-lined pond.

In this work, the impact of initial pH on the production of volatile fatty acids (VFAs) of hybrid Pennisetum was investigated. The batch experiments were conducted under six distinct beginning pH at a mesophilic temperature. Initial pH had an obvious effect on VFA yield and composition, and severe alkaline circumstances (pHin = 11.0) could boost VFA production and acetic acid selectivity. The highest VFAs yield and acetate proportion were obtained when the initial pH was 11.0, with 518 ± 29 mg g−1VS and 92%. Furthermore, microbial community analysis showed that alkaliphilic acetogenic anaerobe such as Amphibacillus, Tissierella, and Natronincola were the dominant species when the initial pH was 11.0. The Amphibacillus is the main hydrolysis bacterium under these conditions because of its high ability for xylan degradation at pH 9.0–10.0. Because of the increased VFA yield and superior acetic acid selectivity, the results suggest that adjusting the initial pH to 11.0 in batch mode would be possible for scaling-up purposes.

Constituents of the seed microbiota and initial changes in the microbiota in fermentations are important in fermentation progression. To identify the origin of indigo-reducing bacteria and understand the initial changes in the microbiota that occur concomitantly with the initiation of indigo reduction during indigo fermentation, we analysed the initial changes in the microbiota. The proportions of the reported indigo-reducing taxa Alkalibacterium, Amphibacillus and Polygonibacillus increased to 24.0% on the 5th day, to 15.2% on the 7th day and to 42.8% at 4.5 months, and the relative abundances of these taxa were 0.048%, 0.14% and 0.02%, respectively, in sukumo (composted Japanese indigo plant material used for fermentation). In the early phase of the microbiota transition, two substantial changes were observed. The first change may be attributed to the substantial environmental changes caused by the introduction of heated wood ash extract (pH ≥ 10.5, temperature ≥ 60 °C). This change increased the proportions of Alkalibacterium and the family Bacillaceae. The second change in microbiota might be initiated by the consumption of oxygen by aerobic microorganisms until the 5th day followed by an increase in the abundance of the obligate anaerobe Anaerobranca and the aerotolerant Amphibacillus and a decrease in the abundance of Bacillaceae. This experiment demonstrated that the 0.048% Alkalibacterium in the original material was augmented to 23.6% of the microbiological community within 5 days. This means that using the appropriate material and performing appropriate pretreatment and adjustment of fermentation conditions are important to increase the abundance of the taxa that reduce indigo.

The volatile fatty acids (VFAs) accumulation pattern and microbial community succession were studied during excess sludge (ES) alkaline fermentation at pH of 10.0 with expanded granular sludge blanket reactor over 5 cyclers. Microbial community shifted conspicuously as ES suffered alkaline fermentation. Both VFAs and acid-producing bacteria increased rapidly during the first 8 days fermentation time, and they showed a quite positive correlation relationship. In addition, soluble chemical oxygen demand (SCOD) also dramatically increased during the first 8 days, which implied 8 day was the optimum sludge retention time (SRT) for ES alkaline fermentation and VFAs accumulation time. Illumina Miseq Sequencing analysis indicated that Clostridium , Bacillus , Amphibacillus and Peptostreptococcaceae were the dominant bacteria genus to produce VFAs. Acetic acid took about 84% in total VFAs because among the total acid-producing bacteria most bacteria could produce acetic acid.

© 2015 Elsevier Ltd. The presence and enumeration of halophilic and alkaliphilic bacteria in Spanish-style table-olive fermentations was studied. Twenty 10-tonne fermenters at two large manufacturing companies in Spain, previously studied through both culture dependent and independent (PCR-DGGE) methodologies, were selected. Virtually all this microbiota was isolated during the initial fermentation stage. A total of 203 isolates were obtained and identified based on 16S rRNA gene sequences. They belonged to 13 bacterial species, included in 11 genera. It was noticeable the abundance of halophilic and alkaliphilic lactic acid bacteria (HALAB). These HALAB belonged to the three genera of this group: Alkalibacterium, Marinilactibacillus and Halolactibacillus. Ten bacterial species were isolated for the first time from table olive fermentations, including the genera Amphibacillus, Natronobacillus, Catenococcus and Streptohalobacillus. The isolates were genotyped through RAPD and clustered in a dendrogram where 65 distinct strains were identified. Biodiversity indexes found statistically significant differences between both patios regarding genotype richness, diversity and dominance. However, Jaccard similarity index suggested that the halophilic/alkaliphilic microbiota in both patios was more similar than the overall microbiota at the initial fermentation stage. Thus, up to 7 genotypes of 6 different species were shared, suggesting adaptation of some strains to this fermentation stage. Morisita-Horn similarity index indicated a high level of codominance of the same species in both patios. Halophilic and alkaliphilic bacteria, especially HALAB, appeared to be part of the characteristic microbiota at the initial stage of this table-olive fermentation, and they could contribute to the conditioning of the fermenting brines in readiness for growth of common lactic acid bacteria., This research was funded by the Spanish Ministry of Science and Innovation (MICINN), through Projects AGL2009-07861 and AGL2012-33400, and by the Junta de Andalucía Excellence Projects AGR-04621 and AGR-07345. All these projects included FEDER funds. AMB was the recipient of a post-doctoral grant awarded by the Junta de Andalucía as part of the Project AGR-07345. HLP was the recipient of a contract funded by the Spanish Ministry of Economy and Competitiveness as part of the Project AGL2012-33400. We want to express our most sincere gratitude to Juan Carlos Roldán, from JOLCA S.A., and Antonio Martín and Marta Sánchez, from GOYA en España S.A.U., for their invaluable collaboration in this study.

A Gram-stain-negative, non-motile, strictly aerobic bacterium designated BW11-2T was isolated from marine sediment of the south-west Indian Ocean. Cells of BW11-2T were rod-shaped, endospore-forming, 0.3–0.5 µm wide, 1.8–2.0 µm long, catalase-positive and oxidase-negative. The isolate was capable of growing at 15–45 °C (optimum 30 °C), pH 5–9 (optimum 7) and with 0.5–10 % (w/v) NaCl (optimum 3 %). Based on 16S rRNA gene sequence similarities, BW11-2T was shown to belong to the family Bacillaceae within the phylum Firmicutes and formed a distinct lineage, showing the highest sequence similarities to closely related genera: Bacillus (93.9–94.7 %), Graci libacillus (93.3–93.7 %), Amphibacillus (93.5 %), Virgibacillu s (92.9–93.1 %) and Anaerobacillus (92.6–93.0 %). BW11-2T shared the highest 16S rRNA gene sequence similarity with the species Bacillus oleronius (94.7 %). The predominant fatty acids (>10 %) were anteiso-C15 : 0 and iso-C15 : 0. The major quinone was menaquinone-7 (MK-7). Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and one unidentified aminolipid. The genomic DNA G+C content of strain BW11-2T was 43.3 mol%. On the basis of the morphological and chemotaxonomic characteristics as well as genotypic data, strain BW11-2T represents a novel genus and species in the family Bacillaceae , for which the name Swionibacillus sediminis gen. nov., sp. nov. is proposed. Strain BW11-2T (=CICC 24196T=JCM 31924T) is the type strain.

Although the indigo reduction process is performed via natural fermentation and maintained under open-air condition, the indigo-reducing reactions continue for 6 months (on average) or longer. Identifying the mechanism underlying the maintenance of this process could lead to the development of a novel, long-lasting, unsterilized bioprocesses. To determine the mechanisms underlying the maintenance of the indigo fermentation system microbiota for more than 6 months in a reduced state in an anaerobic alkaline environment, we examined changes in the microbiota in one early-phase batch and two aged batches of indigo fermentation fluid. The microbiota in the aged fermentation fluid consisted mainly of the genera Alkalibacterium, Amphibacillus, Anaerobacillus and Polygonibacillus and the family Proteinivoraceae. The genera Alkalibacterium, Amphibacillus and Polygonibacillus are known to include indigo-reducing bacteria. Although the transition speed was slower in the aged fermentation fluid than in the early-stage fluid, the microbiota in the aged fermentation fluid maintained for more than 6 months was drastically changed within a period of 3 months. The results of this study indicate that the bacterial consortia consisted of various indigo-reducing species that replace the previous group of indigo-reducing bacteria. The notable transitional changes may be concomitant with changes in the environmental conditions, such as the nutritional conditions, observed over 3 months. This flexibility may lead to important changes in the microbiota that allow for the maintenance of a fermentation-reducing state over a long period.

The genus Amphibacillus was established in 1990, and seven additional species were described in the past two decades. Amphibacillus jilinensis Y1(T) is a facultatively anaerobic and alkaliphilic bacterium isolated from a soda lake in China. Here we describe the structural and genetic features of the draft genome about the type strain Y1(T) (3,831,075 bp, with a G+C content of 37.27%). This is the first genome report of the Amphibacillus genus.

Indigo fermentation has been traditionally performed for dyeing textiles in Japan. Limited information is available on the microbiota involved and the succession of the bacterial community structure during indigo reduction. We investigated the bacterial community structure associated with indigo fermentation using denaturing gradient gel electrophoresis and clone library analyses of a PCR-amplified 16S rRNA gene in the early phase of fermentation carried out in our laboratory. A marked substitution of Halomonas spp. by Amphibacillus spp. was observed corresponding to the marked change in the state of indigo reduction. Although the reported indigo-reducing bacteria, Alkalibacterium spp., were not predominant in the early phase of fermentation, they were predominant in fermentation liquor aged for 10 months obtained from Date City, Japan, as determined by culture-dependent and culture-independent analyses. Novel indigo-reducing strains, Amphibacillus spp. strain C40 and Oceanobacillus spp. strain A21, were isolated from fermentation liquor aged for 10 months and from liquor aged for 4 days, respectively. It is considered that, in addition to the strains belonging to the genus Alkalibacterium, strains belonging to genera Amphibacillus and Oceanobacillus play important roles in sustaining the reduced state of indigo during fermentation.

Culture-independent studies show that soda lake environments harbour diverse groups of bacteria and archaea. In this study different enrichment and isolation media were used in an attempt to isolate novel groups of bacteria from Lake Elmenteita. Different media were prepared using filter-sterilised water from the lake. The isolates recovered were purified on tryptic soy agar supplemented with 1% sodium carbonate and 4% sodium chloride. Phylogenetic analysis of 181 partial 16S rRNA gene sequences with excellent quality showed that the majority of the isolates were affiliated to the class Gammaproteobacteria and to the genus Bacillus. Isolates from the genus Halomonas and Bacillus constituted 37 and 31% of the total sequenced isolates, respectively. Other groups recovered were related to Marinospirillum, Idiomarina, Vibrio, Enterococcus, Alkalimonas, Alkalibacterium, Amphibacillus, Marinilactibacillus and the actinobacteria Nocardiopsis and Streptomyces. Fifty-one different genera were represented with 31 and 15 cultures scoring with their nearest neighbour similarities below 98 and 97%, respectively. Some novel taxa were identified which had not been isolated previously from the soda environment. The results show that the use of different media with varying compositions can help retrieve novel bacterial diversity from the soda lake environment.

Amphibacillus is a genus for Gram-positive, spore-forming, rod-shaped, facultatively anaerobic bacteria with low-G+C content of DNA, established by Niimura et al. in 1990. Amphibacillus xylanus, the type species of the genus, grows well under both strictly anaerobic and aerobic conditions in spite of lacking any isoprenoid quinones, cytochromes, and catalase. Amphibacillus fermentum and Amphibacillus tropicus were later proposed by Zhilina et al. in 2001 for the isolates from a soda lake. In this paper, we revealed the latter two species also lacked isoprenoid quinones, cytochrome and catalase, and that they grew well under strictly anaerobic and aerobic conditions. The consistent growth of A. xylanus under both conditions is due to the presence of anaerobic and aerobic pathways for glucose metabolism in the organism. Although A. fermentum and A. tropicus are supposed to have a side enzymatic pyruvate pathway to produce lactate under both conditions, the two species have two major pyruvate metabolic pathways as observed in A. xylanus. Analysis data indicated that NADH formed both by the aerobic pyruvate pathway and by the glycolytic pathway was re-oxidized by the NADH oxidase in A. fermentum and A. tropicus as well as A. xylanus, and furthermore that the NADH oxidase-Prx (AhpC) system, i.e., NADH oxidase scavenging hydrogen peroxide with Prx, also functions in A. tropicus as observed with A. xylanus. Not only the taxonomical character of the genus Amphibacillus but also the growth characterization based on the two metabolic pathways and unique oxygen metabolism are distinctive in those traits from other facultative anaerobes.

Nitrogen fixation (NF) potential was measured in more than 40 samples of soda solonchak soils with the pH of water extract between 9.5 and 11.0 collected in several locations of Central Asia and in Egypt, using the acetylene reduction method. NF was detected in most of the samples. Maximal rates were observed under microaerophilic–anaerobic conditions with glucose as a substrate. In most cases, the NF negatively correlated with salt content and alkalinity. Five enrichments at pH 10 under micro-oxic conditions with glucose resulted in stable haloalkaliphilic mixed cultures, with diazotrophic component(s) active up to 2.0–3.0 M total Na+. The cultures were dominated by Gram-positive spore-forming bacteria. Molecular cloning of nifH genes demonstrated the presence of two phylogenetic lineages of diazotrophs in the enrichments affiliated with the low-GC Gram-positive bacteria (in rRNA groups 1 and 6 of bacilli and in Clostridiales). Isolation of pure cultures of haloalkaliphilic diazotrophs from micro-oxic enrichments yielded nine strains, comprising two phylogenetic lineages. Most of the isolates (eight) were affiliated with the aerotolerant fermentative haloalkaliphilic bacterium Amphibacillus tropicus and a single strain clustered with the obligately anaerobic haloalkaliphile Bacillus arseniciselenatis. Diazotrophy has never been recognized previously in these groups of Gram-positive bacteria. Overall, the results demonstrated the existence, in soda solonchak soils, of a novel group of free-living fermentative diazotrophic bacteria active at extremely haloalkaline conditions.

Terasi is a traditional product generally made of fermented shrimp. There were many studies regarding lactic acid bacteria of terasi but none regarding proteolitic bacteria. This study was conducted to isolate and identify the thermophilic and mesophylic proteolytic bacteria from terasi. In addition, the effect of different salt concentrations on the growth of the isolated proteolytic bacteria with the greatest proteolytic activity was also studied. Terasi samples were obtained from the Northern coast region of Java island i.e. Jepara, Demak and Batang. The study obtained 34 proteolytic isolates. Four isolates were identified as Sulfidobacillus, three isolates as Vibrio / Alkaligenes / Aeromonas, two isolates as Pseudomonas, 21 isolates as Bacillus, three isolates as Kurthia/ Caryophanon and one isolates as Amphibacillus. The growth of proteolytic bacteria was affected by salt concentration. The largest growth was found at 0 ppm salt concentrations and growth was declined as salt concentration increased. Maximum growth at each salt concentration tested was found at 8 hours incubation.

New alkaliphilic, saccharolytic, rod-shaped, gram-positive bacteria resistant to heating and drying and phylogenetically affiliated to the Bacillus lineage were isolated under strictly anaerobic conditions from sediments of the alkaline and highly mineralized Lake Magadi. Strain Z-7792 forms endospores; in strain Z-7984, endospore formation was not revealed. The strains are capable of both anaerobic growth (at the expense of fermentation of glucose and certain mono- and disaccharides with the formation of formate, ethanol, and acetate) and aerobic growth. Among polysaccharides, the strains hydrolyze starch, glycogen, and xylan. Yeast extract or methionine are required for growth. The strains are strict alkaliphiles exhibiting obligate requirement for Na+ and carbonate ions but not for Cl- ion. Growth occurs at a total mineralization as high as 3.3-3.6 M Na+, with an optimum at 1-1.7 M Na+. Strain Z-7792 is an obligate alkaliphile with a pH growth range of 8.5-11.5 and an optimum of 9.5-9.7. Strain Z-7984 grows in a pH range of 7.0-10.5 with an optimum at 8.0-9.5. Both strains are mesophiles having a growth optimum at 37-38 degrees C. They belong to bacilli with a low G + C content. The G + C contents of the DNA of strains Z-7792 and Z-7984 are 39.2 and 41.5 mol%, respectively. These isolates of facultatively anaerobic, strictly alkaliphilic, Na(+)-dependent bacilli can be considered representatives of the ecological group adapted to the life at drying-up shoars of soda lakes. Because of their independence of NaCl and lack of obligate dependence on sodium carbonates, the isolates are to be assigned to athalassophilic organisms. According to their physiological and phylogenetic characteristics, they taxonomically belong to group 1 of the species of bacilli, occupying a position intermediate between the genera Amphibacillus and Gracilibacillus. The isolates are described as new species of Amphibacillus: A. fermentum (type strain, Z-7984T) and A. tropicus (type strain, Z-7792T).

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H2O2 and exhibited a specific activity of 46 μmol NADH min–1 (mg protein)–1. NADPH did not serve as a substrate for the enzyme. The Km for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl.

Twelve strains named Bacillus pantothenticus, at least 29 Bacillus strains representing 16 species belonging to rRNA groups 1 and 2, one Bacillus dipsosauri strain, and 38 strains of Amphibacillus, Aneurinibacillus, Brevibacillus, Halobacillus, Paenibacillus, Sporosarcina and Marinococcus, were characterized genotypically using amplified rDNA restriction analysis (ARDRA), and phenotypically using routine diagnostic characters comprising 61 biochemical tests in the API System and 15 observations of vegetative cell and sporangial morphology. The B. pantothenticus strains were also characterized by fatty acid methyl ester analysis and SDS-PAGE of whole-cell proteins. ARDRA revealed that strains of B. pantothenticus formed a cluster quite separate from other species in rRNA group 1, supporting the recognition of the former as a separate genus, for which the name Virgibacillus is proposed. The polyphasic data also indicate the presence of an as yet undescribed new species within this genus. The species Virgibacillus pantothenticus and related organisms comprising this new genus can be distinguished from members of Bacillus rRNA group 1 (Bacillus sensu stricto), and from members of Paenibacillus and other aerobic endospore-forming bacteria by routine phenotypic tests.

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 μM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca²⁺ and Mn²⁺ slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 μM Cr(VI) and 72 μmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 μM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.

Amphibacillus NADH oxidase belongs to a growing new family of peroxiredoxin-linked oxidoreductases including alkyl hydroperoxide reductase F (AhpF). Like AhpF it displays extremely high hydroperoxide reductase activity in the presence of a Prx, thus making up the NADH oxidase-Prx system. The NADH oxidase primarily catalyzes the reduction of oxygen by NADH to form H2O2, while the Prx immediately reduces H2O2 (or ROOH) to water (or ROH). Consequently, the NADH oxidase-Prx system catalyzes the reduction of both oxygen and hydrogen peroxide to water with NADH as the preferred electron donor. The NADH oxidase-Prx system is widely distributed in aerobically growing bacteria lacking a respiratory chain and catalase, and plays an important role not only in scavenging hydroperoxides but also in regenerating NAD in these bacteria

Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe3O4) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe3O4 nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)2, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca2+ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology.

Endospore-forming strains were isolated from corn-compost treated with olive-mill wastewater ('alpechin'). The strains were taxonomically studied and proposed as a novel Paenibacillus species. These organisms (strains B.3T, B.7 and B.9) were particularly distinguishable from other aerobic spore-forming species by their ability to grow optimally in 100% (v/v) olive-mill wastewater at 30 degrees C and pH 7.0 and concomitant production of an interesting exopolysaccharide. Chemotaxonomic analysis revealed that MK-7 was the predominant menaquinone, the major fatty acid was anteiso C15:0 and the cell wall contained meso-diaminopimelic acid. The DNA G+C content was 40.7 mol%. Comparative sequence analysis of 16S rDNA with different reference species from the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Alicyclobacillus, Halobacillus, Virgibacillus, Amphibacillus, Coprobacillus and Gracilibacillus indicated that the isolated strains were highly related to the genus Paenibacillus. Strain B.3T formed an evolutionary lineage distinct from other species within the evolutionary radiation encompassing the genus Paenibacillus. Strain B.3T was a close relative of Paenibacillus polymyxa, but DNA-DNA relatedness data with this species was very low (relative binding ratio < 16%). Based on the morphological and physiological characteristics, as well as on the phylogenetic position determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these strains should be designated a novel species, for which the name Paenibacillus jamilae sp. nov. is proposed. The type strain is B.3T (= CECT 5266T = DSM 13815T).

It was found that a facultatively anaerobic and halophilic alkaliphile, M-12 (Amphibacillus sp.), possesses a Na(+)-stimulated ATPase in the membrane. The ATPase activity was inhibited by NO3- and SCN- which are the inhibitors of V-type ATPase, but not by azide and vanadate, inhibitors of F-type ATPase and P-type ATPase, respectively. Upon the incubation of the membrane in buffer containing ATP and MgCl2, several polypeptides were released from the membrane. Among them, two major polypeptides with apparent molecular masses of 79 and 55 kDa crossreacted with an antiserum against the catalytic units (subunits A and B) of V-type ATPase from Enterococcus hirae. The N-terminal amino acid sequences of the 79 and 55 kDa polypeptides showed high similarity to those of subunits A and B of V-type ATPase from Enterococcus hirae, respectively. M-12 is likely to possess a V-type Na(+)-ATPase.

16S rRNA gene sequences of the type strains of 11 species belonging to the Bacillus brevis and Bacillus aneurinolyticus groups were determined. On the basis of the results of gene sequence analyses, these species were separated into two clusters. The B. brevis cluster included 10 species, namely, Bacillus brevis, Bacillus agri, Bacillus centrosporus, Bacillus choshinensis, Bacillus parabrevis, Bacillus reuszeri, Bacillus formosus, Bacillus borstelensis, Bacillus laterosporus, and Bacillus thermoruber. Bacillus aneurinolyticus and Bacillus migulanus belonged to the B. aneurinolyticus cluster. Moreover, the two clusters were phylogenetically distinct from other Bacillus, Amphibacillus, Sporolactobacillus, Paenibacillus, and Alicyclobacillus species. On the basis of our data, we propose reclassification of the B. brevis cluster as Brevibacillus gen. nov. and reclassification of the B. aneurinolyticus cluster as Aneurinibacillus gen. nov. By using 16S rRNA gene sequence alignments, two specific PCR amplification primers were designed for differentiating the two new genera from each other and from other aerobic, endospore-forming organisms.

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Amphibacillus xylanus DSM 6626 was enriched 100-fold to homogeneity. The molecular mass was determined by native polyacrylamide electrophoresis and by gel filtration to be 260 kDa (+/- 25 kDa); the enzyme was composed of identical subunits of 45 (+/- 5) kDa, indicating that the native enzyme has a hexameric structure. NAD-GDH was highly specific for the coenzyme NAD(H) and catalyzed both the formation and the oxidation of glutamate. Apparent Km-values of 56 mM glutamate, 0.35 mM NAD (oxidative deamination) and 6.7 mM 2-oxoglutaric acid, 42 mM NH4Cl and 0.036 mM NADH (reductive amination) were measured. The enzyme was unusually resistant towards variation of pH, chaotropic agents, organic solvents, and was stable at elevated temperature, retaining 50% activity after 120 min incubation at 85 degrees C.

Three strains of gram-positive, facultatively anaerobic, sporeforming, rod-shaped bacteria were isolated from composts of manure with grass and rice straw. These organisms grew well in an alkaline medium and digested xylan both in strictly anaerobic cultures when titanium(III) citrate was used as a reducing agent and in aerobic cultures with shaking. The cells contained meso-diaminopimelic acid, and their cellular fatty acids consisted of iso- and anteiso-branched acids and considerable amounts of straight-chain acids. The DNA base composition of these strains ranged from 36 to 38 mol% guanine plus cytosine. Cytochromes, isoprenoid quinones, and catalase activity were not detected. DNA-DNA homology determinations did not show relatedness to strains of representative species of the genera Bacillus, Clostridium, and Sporolactobacillus. Considering the uniqueness of the characteristics, the sequence of the 5S rRNA, and the unique metabolic pathways, we propose Amphibacillus xylanus gen. nov., sp. nov., for these strains. The type strain is strain Ep01 (= JCM 7361).

A strain KSUCr3 with extremely high Cr(VI)-reducing ability under alkaline conditions was isolated from hypersaline soda lakes and identified as Amphibacillus sp. on the basis of 16S rRNA gene sequence analysis. The results showed that Amphibacillus sp. strain KSUCr3 was tolerant to very high Cr(VI) concentration (75 mM) in addition to high tolerance to other heavy metals including Ni 2+ (100 mM), Mo 2+ (75 mM), Co 2+ (5 mM), Mn 2+ (100 mM), Zn 2+ (2 mM), Cu 2+ (2 mM) and Pb (75 mM). Strain KSUCr3 was shown to be of a high efficiency in detoxifying chromate, as it could rapidly reduce 5 mM of Cr(VI) to a non detectable level over 24 hrs. In addition, strain KSUCr3 could reduce Cr(VI) efficiently over a wide range of initial Cr(VI) concentrations (1-10 mM) in alkaline medium under aerobic conditions without significant effect on the bacterial growth. Addition of glucose, NaCl and Na 2 CO 3 to the culture medium caused a dramatic increase in Cr(VI)-reduction by Amphibacillus sp. strain KSUCr3. The maximum chromate removal was exhibited in alkaline medium containing 1.5% Na 2 CO 3 , 0.8% glucose, and 1.2% NaCl, at incubation temperature of 40oC and shaking of 100 rpm. Under optimum Cr(VI) reduction conditions, Cr(VI) reduction rate reached 237 µMh 1 which is one of the highest Cr(VI) reduction rate, under alkaline conditions and high salt concentration, compared to other microorganisms that has been reported so far. Furthermore, the presence of other metals, such as Ni 2+ , Co 2+ , Cu 2+ and Mn 2+ slightly stimulated Cr(VI)-reduction ability by the strain KSUCr3. The isolate, Amphibacillus sp. strain KSUCr3, exhibited an ability to repeatedly reduce hexavalent chromium without any amendment of nutrients, suggesting its potential application in continuous bioremediation of Cr(VI). The results also revealed the possible isolation of potent heavy metals resistant bacteria from extreme environment such as hypersaline soda lakes.