An Endophytic <Emphasis Type="Italic">Phomopsis</Emphasis> sp. Possessing Bioactivity and Fuel Potential with its Volatile Organic Compounds

An unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), associated with a cloud forest in Northern Ecuador. This fungus produces a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor) only previously known from higher plants. In addition, some of the other more abundant VOCs recorded by GC/MS in this organism were 1-butanol, 3-methyl; benzeneethanol; 1-propanol, 2-methyl and 2-propanone. The gases of Phomopsis sp. possess antifungal properties and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including: Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. The IC₅₀ values for the artificial gas mixture of Phomopsis sp. varied between 8 and 25.65 μl/mL. Proton transfer reaction-mass spectrometry monitored the concentration of VOCs emitted by Phomopsis sp. and yielded a total VOC concentration of ca. 18 ppmv in the head space at the seventh day of incubation at 23°C on PDA. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus. A discussion is presented on the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship.     

Bioprocessing of agricultural residues to ethanol utilizing a cellulolytic extremophile

A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70°C and 15–20% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.45–0.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.     

Case study of a biological control: <Emphasis Type="Italic">Geobacillus caldoxylosilyticus</Emphasis> (IRD) contributes to alleviate salt stress in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants

The inevitable exposure of crop plants to salt stress is a major environmental problem emerged from the presence of excess NaCl radicals in the soil. Handling the problem in maize plants using a biological agent was the main interest of the present study. The non-pathogenic, halophytic, facultative aerobic bacterium Geobacillus caldoxylosilyticus IRD that was isolated from Marakopara pond in the Atoll Tikehau (French Polynesian, 2005) and found tolerant to salt stress until 3.5% NaCl (w/v). An artificial symbiosis was achieved by inoculating Geobacillus sp. into 5-day-old maize cultivars of triple hybrids (321 and 310) and singlet hybrids (10 and 162). Thereafter, maize seedlings were exposed to 350 mmol NaCl for 10 days. The data revealed that Geobacillus sp. had interacted with salinized maize and improved maize overall growth, dry weight and relative water content. Na⁺ accumulation was six times less and Cl⁻ accumulation was 13 times less in the tips of salinized maize seedlings upon Geobacillus sp. inoculation. Salinized maize without Geobacillus viewed decayed cortical cells of seedlings. In addition, proline content was two times higher in salinized seedlings lacking Geobacillus. Photosynthetic pigments and antioxidant enzymes were significantly regulated upon inoculation. Beyond this study, we presented a novel insight into a possible role of Geobacillus caldoxylosilyticus bacteria in controlling/protecting maize plants against high salt stress.     

Phylogenetic,Inter, and Intraspecific Sequence Analysis of <Emphasis Type="Italic">spo0A</Emphasis> Gene of the Genus <Emphasis Type="Italic">Geobacillus</Emphasis>

In this work, the variability of spo0A gene in the genus Geobacillus and applicability of this gene for the taxonomy within this genus were evaluated. The protein Spo0A is the master regulator of the endospore-forming process in the all endospore-forming bacteria. Geobacillus genus-specific primers GEOSPO were designed based on the sequences of Geobacillus spo0A gene available through the public databases. Inter and intraspecific variability of Geobacillus spo0A gene was determined after sequencing of the GEOSPO-PCR products. Geobacillus spo0A sequence analysis showed that three species—Geobacillus thermodenitrificans, G. stearothermophilus, and G. jurassicus—could be easily identified. Similarity between the sequences of these species and the other species were in the range of 83.3%–92.0%. In contrast, intraspecific similarity of G. thermodenitrificans and G. stearothermophilus was high—above 99.0%. Similarity of spo0A sequences of G. subterraneus–G. uzenensis species cluster also matched this interval. Intercluster similarity between G. lituanicus–G. thermoleovorans–G. kaustophilus–G. vulcani and G. thermocatenulatus–G. gargensis–G. caldoxylosilyticus–G. toebii–G. thermoglucosidasius species clusters, as well as interspecific similarity within these two clusters was in the range of the intraspecific similarity determined for G. thermodenitrificans and G. stearothermophilus. It was also determined that spo0A cannot be used as the phylogenetic marker for the genus Geobacillus.     

<Emphasis Type="Italic">Scopulibacillus darangshiensis</Emphasis> gen. nov., sp. nov., isolated from rock

A novel, Gram-positive bacterium, designated DLS-06^T, was isolated from scoria (volcanic ash) under rock on the peak of small mountain (300 m above the sea level; known as Darangshi Oreum) in Jeju, Republic of Korea. The cells of the isolate were aerobic, oxidase-negative, catalase-positive, endospore-forming, non-motile rods. The organism grew at 25∼30°C and initial pH 6.1∼9.1. A neighbour-joining tree based on 16S rRNA gene sequences showed that the organism was related to members of the family “Sporolactobacillaceae” and related taxa. The phylogenetic neighbours were Pullulanibacillus naganoensis (95.2% 16S rRNA gene sequence similarity), Tuberibacillus calidus (95.0%) and Sporolactobacillus (91.8∼94.2%). Levels of 16S rRNA gene sequence similarity of the isolate to representatives of other genera were in the range of 87.2∼93.7%. The organism contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The predominant menaquinone was MK-7. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, an unknown ninhydrin-positive phospholipid, three unknown phospholipids and an unknown lipid. The major fatty acids were anteiso-C_15:0 and anteiso-C_17:0. The G+C content of the DNA was 50.8 mol%. On the basis of the phenotypic and phylogenetic data presented in this study, this organism represents a novel genus and species in the order Bacillales, for which the name Scopulibacillus darangshiensis gen. nov., sp. nov. is proposed. The type strain is DLS-06^T (=DSM 19377^T =KCTC 13161^T).     

Janibacter alkaliphilus sp. nov., isolated from coral Anthogorgia sp

A novel actinobacterium, designated strain SCSIO 10480^T, was isolated from a gorgonian coral sample of Anthogorgia sp. Phylogenetic and phenotypic properties of the organism supported that it belonged to the genus Janibacter. Phylogenetic analysis indicated that the levels of 16S rRNA gene sequence similarity between strain SCSIO 10480^T and other type strains of recognized members of the genus Janibacter were 96.0–97.8 %. Growth in the presence of up to 17 % (w/v) NaCl and optimally at pH 9.0–10.0 was a distinctive characteristic of strain SCSIO 10480^T. Other biochemical and physiological properties and the fatty acid profile also differentiated the isolate from other members of Janibacter species. Based on the results obtained in this study, we propose that strain SCSIO 10480^T should be classified within a novel species of the genus Janibacter, for which the name Janibacter alkaliphilus sp. nov. is proposed, with SCSIO 10480^T (=CCTCC AB 2011027^T = DSM 24723^T) as the type strain.     

Cloning and Characterization of a 4-Hydroxyphenylacetate 3-Hydroxylase From the Thermophile <Emphasis Type="Italic">Geobacillus</Emphasis> sp. PA-9

A 4-hydroxyphenylacetic acid (4-HPA) hydroxylase-encoding gene, on a 2.7-kb genomic DNA fragment, was cloned from the thermophile Geobacillus sp. PA-9. The Geobacillus sp. PA-9 4-HPA hydroxylase gene, designated hpaH, encodes a protein of 494 amino acids with a predicted molecular mass of 56.269 Da. The deduced amino-acid sequence of the hpaH gene product displayed <30% amino-acid sequence identity with the larger monooxygenase components of the previously characterized two-component 4-HPA 3-hydroxylases from Escherichia coli W and Klebsiella pneumoniae M5a1. A second oxidoreductase component was not present on the 2.7-kb genomic DNA fragment. The deduced amino-acid sequence of a second C-terminal truncated open reading frame, designated hpaI, exhibited homology to extradiol oxygenases and displayed the highest amino-acid sequence identity (43%) with the 3,4-dihydroxyphenylacetate 2,3-dioxygenase of Arthrobacter globiformis, encoded by mndD. These results, along with catalytic activity observed in crude intracellular extracts prepared from Escherichia coli cells expressing hpaH, is in support of a role for hpaH in the 4-HPA degradative pathway of Geobacillus sp. PA-9.     

Isolations of α-glucosidase-producing thermophilic bacilli from hot springs of Turkey

From 42 different hot springs in six provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the α-glucosidase production capacities by using pNPG as a substrate. α-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced α-glucosidase at significant levels comparable with that of the reference strains tested; thus, five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g), were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes in α-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to α-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a, and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular α-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in α-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable α-1,4-glucosidase-producing bacilli that have biotechnological potential with α-glucosidases located at different cell positions were obtained. The text was submitted by the authors in English.     

Lysinibacillus mangiferahumi sp. nov., a new bacterium producing nematicidal volatiles

A Gram-positive bacterium, designated M-GX18^T, was isolated from the rhizosphere soil of mango (Guangxi Province, China). The isolate produced nematicidal volatile compounds with activities against the root-knot nematode Meloidogyne incognita. The organism was motile, catalase- and oxidase-positive, spore-forming and rod shaped. The predominant menaquinone was MK-7 and the major cellular fatty acid profiles were iso-C_15:0, anteiso-C_15:0 and iso-C_16:0. The DNA G+C content was 38.9%. A phylogenetic analysis based on 16S rRNA gene sequences showed that this organism represented a new species of the genus Lysinibacillus. Strain M-GX18^T exhibited high 16S rRNA gene sequence similarity with its closest neighbors Lysinibacillus sphaericus (98.5%), Lysinibacillus fusiformis (98.1%) and Lysinibacillus xylanilyticus (98.6%). The physiological, biochemical and chemotaxonomic data, including DNA–DNA hybridization relatedness data, indicate that strain M-GX18^T can be distinguished from all the related species of the genus Lysinibacillus. Therefore, on the basis of the polyphasic taxonomic data presented, a new species of the genus Lysinibacillus, Lysinibacillus mangiferahumi, with the type strain M-GX18^T (=DSM 24076^T = CCTCC AB 2010389^T) is proposed.     

<Emphasis Type="Italic">Geobacillus zalihae</Emphasis> sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

Background  

Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5–99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp. strain: purification and properties

The purpose of this study was purification and characterization of catechol 1,2-dioxygenase from Geobacillus sp. G27 strain, which degrades α-naphthol by the β-ketoadipate pathway. The catechol 1,2-dioxygenase (C1,2O) was purified using four steps of ammonium sulfate precipitation, DEAE-celullose, Sephadex G-150 and hydroxylapatite chromatographies. The enzyme was purified about 18-fold with a specific activity of 7.42 U mg of protein⁻¹. The relative molecular mass of the native enzyme estimated on gel chromatography of Sephadex G-150 was 96 kDa. The pH and temperature optima for enzyme activity were 7 and 60°C, respectively. A half-life of the catechol 1,2-dioxygenase at the optimum temperature was 40 min. The kinetic parameters of the Geobacillus sp. G27 strain catechol 1,2-dioxygenase were determined. The enzyme had apparent K_m of 29 μM for catechol and the cleavage activities for methylcatechols were much less than for catechol and no activity with gentisate or protocatechuate was detected.     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

Cloning,purification and characterization of an alkali-stable endoxylanase from thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 71

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al³⁺ and Cu²⁺ but strongly inhibited by Hg²⁺. The enzyme follows Michaelis–Menten kinetics, with K_m and V_max values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.     

Virgibacillus zhanjiangensis sp. nov., a marine bacterium isolated from sea water

A Gram-positive, endospore-forming, catalase- and oxidase-positive, motile, rod-shaped, aerobic bacterium, designated strain JSM 079157^T, was isolated from surface seawater off the coastline of Naozhou Island in South China Sea. The organism was able to grow with 1–15% (w/v) total salts (optimum, 4–7%), and at pH 6.0–10.0 (optimum, pH 7.5) and 10–45°C (optimum, 30°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7, and the polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids were anteiso-C_15:0 (45.1%) and anteiso-C_17:0 (16.2%), and the DNA G + C content was 39.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 079157^T should be assigned to the genus Virgibacillus, being related most closely to the type strains of Virgibacillus litoralis (97.4% sequence similarity), Virgibacillus necropolis (97.3%) and Virgibacillus carmonensis (97.1%). These four strains formed a distinct subcluster in the phylogenetic tree. The levels of DNA–DNA relatedness between the new isolate and the type strains of V. litoralis, V. necropolis and V. carmonensis were 30.4, 19.3 and 12.6%, respectively. The results of the phylogenetic analysis, combined with DNA–DNA relatedness data, phenotypic characteristics and chemotaxonomic information, support the suggestion that strain JSM 079157^T represents a new species of the genus Virgibacillus, for which the name Virgibacillus zhanjiangensis sp. nov. is proposed. The type strain is JSM 079157^T (=DSM 21084^T = KCTC 13227^T).     

Hydrophobic response of the fungus <Emphasis Type="Italic">Rhinocladiella similis</Emphasis> in the biofiltration with volatile organic compounds with different polarity

Rhinocladiella similis biodegraded volatile organic compounds (VOCs) of different polarity in gas-phase biofilters. Elimination capacities, (EC) of 74 g_hexane m⁻³ h⁻¹, 230 g_ethanol m⁻³ h⁻¹, 85 g_toluene m⁻³ h⁻¹ and 30 g_phenol m⁻³ h⁻¹ were obtained. EC values correlated with the solubility of the VOCs. R. similis grown with n-hexane or ethanol in biofilters packed with Perlite showed that the surface hydrophobicity was higher with n-hexane than ethanol. The hydrophobin-like proteins extracted from the mycelium produced with n-hexane (15 kDa) were different from those in the ethanol biofilter (8.5 kDa and 7 kDa).     

New species of sap beetles (Coleoptera: Nitidulidae: Nitidulini) from the Baltic and Bitterfeld ambers

A new genus, Microsoronia, gen. nov., and new species of this genus, M. hoffeinsorum, sp. nov. from the Bitterfeld amber and M. kerneggeri sp. nov., M. nigerrima sp. nov., and M. interfax, sp. nov. from the Baltic amber, are described. The earliest known member of the genus Phenolia, P. (Lasiodites) angustitibialis, sp. nov., is described from the Baltic amber. The systematic position of these two genera, their possible evolution, as well as the possible ecology and bionomics of their members are discussed. It is shown that “Phenolia” incapax Scudder, 1890 should be included in the family Peltidae, rather than Nitidulidae.