Thermoacidophilic Bacterium Research Articles

Biochemical characterization of a thermostable l-arabinose isomerase from a thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68

Abstract The rare monosaccharide d -tagatose is a low-calorie sugar-substituting sweetener, having 92% of relative sweetness but only 1/3 of energy content of sucrose. Industrial production of d -tagatose is carried out from d -galactose by l -arabinose isomerase ( l -AI). It is generally recognized that commercial l -AI for d -tagatose production requires two important enzymatic properties, thermostability and slightly acidic pH optimum. In this article, a thermostable l -AI was characterized from a novel thermoacidophilic bacterium, Alicyclobacillus hesperidum URH17-3-68, which showed promising thermostability and displayed a relatively wide pH spectrum. The araA gene encoding the Al. hesperidum l -AI was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and ion-exchange chromatography. The enzyme displayed maximal activity at 70 °C and pH 7.0, and showed more than 75% of maximal activity from pH 5.5 to 7.0. Cobalt ion was required as optimum metal cofactor for both activity simulation and thermostability improvement at high temperature. The enzyme retained 93% and 63% of initial activity after 4 and 16 h of incubation, respectively, at 75 °C in the presence of Co2+. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate d -galactose were measured to be 54.7 mM, 68.0 min−1, and 1.2 mM−1 min−1, respectively. The bioconversion yield of d -tagatose by the purified enzyme after 27 h at 70 °C reached 43% and 22%, from 50 and 200 mM of d -galactose, respectively. Due to the promising thermostability and high activity at slight acidic pH, the Al. hesperidum l -AI was appropriate for use as a new source of d -tagatose producing enzyme.

Effect of Storage Conditions on Microbiological and Physicochemical Parameters of Cloudy Apple Juice Concentrate

Abstract The effects on microbiological parameters of minimally pasteurized cloudy apple juice concentrate obtained in a pilot scale from two apple varieties, and their changes with storage time and temperature were investigated. The juice obtained from Red Delicious apples showed an adequate biological stability for import and export market for storage at −8.5 and −20°C during a period of three months. The preservation of samples at −20 and −8.5°C limited to acceptable values the growth of molds and yeasts during the first two months in the case of juice obtained from Granny Smith apples. Neither lactic acid nor thermo acidophilic bacteria were isolated from the samples in all storage conditions. No noticeable differences in pH and soluble solids content were observed among storage times and temperatures. Turbidity increased slightly with storage time. Viscosity decreased substantially during storage at 2°C. Color was stable for all storage conditions evaluated.

Draft Genome Sequence of Alicyclobacillus hesperidum Strain URH17-3-68

Alicyclobacillus hesperidum is a thermoacidophilic bacterium. We isolated strain URH17-3-68 from hot spring sludge in Tengchong, Yunnan province, China. Its extracellular products include heat- and acid-stable enzymes which are important for industrial applications. Here we report the draft genome of this strain.

Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

BackgroundThe Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process.ResultsWe report that purified β-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust) were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β-galactosidase molecules present in the adsorption reaction.ConclusionOur results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure. Although the molecular details of spore adsorption have not been fully elucidated, the efficiency of the process and the pH-stability of the adsorbed molecules, together with the well documented robustness and safety of spores of B. subtilis, propose the spore as a novel, non-recombinant system for enzyme display.

A comparison of bioleaching ability of mesophilic and moderately thermophilic culture on copper bioleaching from flotation concentrate and smelter dust

This study evaluates bioleaching treatments of chalcopyrite concentrate and smelter dust using mesophilic and moderately thermophilic bacterial cultures. Effects of some variable parameters such as solids concentration, temperature (type of bacteria) and inoculation on the bioleaching of the materials obtained from Sarcheshmeh copper complex were investigated. Bioleaching experiments were carried out in shaking incubator using mixed mesophilic and moderately thermoacidophilic bacteria. The results indicate that the efficiency of copper extraction is dependent on all the variables studied for both microorganisms. Maximum copper recovery from concentrate and dust was achieved using a moderately thermophilic culture. Under optimum conditions, copper dissolution from concentrate reached 87.52% with moderately thermophilic, and 34.55% with mesophilic culture, after 25 days. Also, overall copper extraction calculated for dust bioleaching were 92.00% with moderately thermophilic bacteria and 73.27% with mesophilic bacteria.

Complete Genome Analysis of Sulfobacillus acidophilus Strain TPY, Isolated from a Hydrothermal Vent in the Pacific Ocean

Sulfobacillus acidophilus strain TPY is a moderately thermoacidophilic bacterium originally isolated from a hydrothermal vent in the Pacific Ocean. Ferrous iron and sulfur oxidation in acidic environments in strain TPY have been confirmed. Here we report the genome sequence and annotation of the strain TPY, which is the first complete genome of Sulfobacillus acidophilus.

Hippea jasoniae sp. nov. and Hippea alviniae sp. nov., thermoacidophilic members of the class Deltaproteobacteria isolated from deep-sea hydrothermal vent deposits

Thirteen novel, obligately anaerobic, thermoacidophilic bacteria were isolated from deep-sea hydrothermal vent sites. Four of the strains, designated EP5-r(T), KM1, Mar08-272r(T) and Mar08-368r, were selected for metabolic and physiological characterization. With the exception of strain EP5-r(T), all strains were short rods that grew between 40 and 72 °C, with optimal growth at 60-65 °C. Strain EP5-r(T) was more ovoid in shape and grew between 45 and 75 °C, with optimum growth at 60 °C. The pH range for growth of all the isolates was between pH 3.5 and 5.5 (optimum pH 4.5 to 5.0). Strain Mar08-272r(T) could only grow up to pH 5.0. Elemental sulfur was required for heterotrophic growth on acetate, succinate, Casamino acids and yeast extract. Strains EP5-r(T), Mar08-272r(T) and Mar08-368r could also use fumarate, while strains EP5-r(T), KM1 and Mar08-272r(T) could also use propionate. All isolates were able to grow chemolithotrophically on H(2), CO(2), sulfur and vitamins. Phylogenetic analysis of 16S rRNA gene sequences placed all isolates within the family Desulfurellaceae of the class Deltaproteobacteria, with the closest cultured relative being Hippea maritima MH(2)(T) (~95-98 % gene sequence similarity). Phylogenetic analysis also identified several isolates with at least one intervening sequence within the 16S rRNA gene. The genomic DNA G+C contents of strains EP5-r(T), KM1, Mar08-272r(T) and Mar08-368r were 37.1, 42.0, 35.6 and 37.9 mol%, respectively. The new isolates differed most significantly from H. maritima MH(2)(T) in their phylogenetic placement and in that they were obligate thermoacidophiles. Based on these phylogenetic and phenotypic properties, the following two novel species are proposed: Hippea jasoniae sp. nov. (type strain Mar08-272r(T) = DSM 24585(T) = OCM 985(T)) and Hippea alviniae sp. nov. (type strain EP5-r(T) = DSM 24586(T) = OCM 986(T)).

Identification of the Medicinal Off-Flavor Compound Formed from Ascorbic Acid and (E)-Hex-2-enal

A test apple beverage made up of apple juice (20%), high-fructose corn syrup (11.5%), citric acid (0.43%), trisodium citrate (0.02%), apple-odor flavor (0.1%), and ascorbic acid (0.02%) was stored at 40 °C and then analyzed for the change of odor in the beverage. Although no thermoacidophilic bacteria (TAB) were detected, a medicinal off-flavor was perceived after the 8 weeks of storage. Model experiments on the ingredients of the test apple beverage revealed that the off-flavor compound had been formed by ascorbic acid and (E)-hex-2-enal. Synthesis and NMR (¹H, ¹³C, HMQC, and HMBC) analyses identified the compound as 6-propylbenzofuran-7-ol. The odor quality, retention index (RI), and mass spectrum of synthetic 6-propylbenzofuran-7-ol were identical with those of the medicinal odor compound from the test apple beverage. Sensory evaluation revealed the recognition thresholds for medicinal odor were 31.4 ppb in water and 24.0 ppb in apple beverage, and the detection thresholds were 19.6 ppb in water and 8.6 ppb in apple beverage, respectively. The quantified concentration of 6-propylbenzofuran-7-ol formed in test apple beverage was 90 ppb, approximately. This concentration was well above the odor threshold, so it was concluded that the compound was the source of the medicinal off-flavor.

Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce

Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60°C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10min at different pressures (100-800MPa) at 40°C. None of these treatments caused any significant inactivation, except perhaps at 800MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300MPa) for A. acidoterrestris and only in a high pressure window (600-800MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800MPa at 25, 40 and 60°C for 10min. At 40°C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60°C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this resulted in up to 3.5 and 2.0 log inactivation for A. acidoterrestris and B. coagulans respectively. We conclude that HP treatment can induce germination and inactivation of spores from thermoacidophilic bacteria in acidic foods, and may thus be useful to reduce spoilage of such foods caused by these bacteria.

Distinctive Responses of Metabolically Active Microbiota to Acidification in a Thermophilic Anaerobic Digester

Acidification is one of the most common and serious problems inducing process failure in anaerobic digesters. The production of volatile fatty acids (VFAs) mainly triggers acidic shock. However, little is known about the bacteria involved in the processes of acidogenic metabolism, such as fermentation and reductive acetogenesis. Here, the metabolic responses of a methanogenic community to the acidification and resulting process deterioration were investigated using transcriptional profiling of both the 16S rRNA and formyltetrahydrofolate synthetase (FTHFS) genes. The 16S rRNA-based analyses demonstrated that the dynamic shift of bacterial populations was closely correlated with reactor performance, especially with VFA accumulation levels. The pH drop accompanied by an increase in VFAs stimulated the metabolic activation of an uncultured Chloroflexi subphylum I bacterium. The subphylum has been characterized as a fermentative carbohydrate degrader using culture- and molecular-based ecophysiological assays. At the beginning of VFA accumulation, FTHFS genes were expressed; the transcripts were derived from phylogenetically predicted homoacetogens, suggesting that reductive acetogenesis was operated by hitherto unidentified bacteria. When acetate concentrations were high, the FTHFS expression ceased and Thermoanaerobacterium aciditolerans proliferated selectively. This thermoacidophilic bacterium would play a decisive role in acetate production via fermentative metabolism. The results of this study reveal for the first time that an uncultured Chloroflexi, T. aciditolerans, and novel homoacetogens were metabolically associated with acidic shock and subsequent VFA accumulation in an anaerobic digester.

Diffuse hydrothermal methane output and evidence of methanotrophic activity within the soils at Sousaki (Greece)

Methane soil flux measurements have been made in 38 sites at the geothermal system of Sousaki (Greece) with the closed chamber method. Fluxes range from )47.6 to 29 150 mg m day, and the diffuse CH4 output of the system has been estimated at 19 t a. Contemporaneous CO2 flux measurements showed a moderate positive correlation between CO2 and CH4 fluxes. Comparison of the CO2 ⁄CH4 soil flux ratios with the CO2 ⁄CH4 ratio of the gases of the main gas manifestations provided evidence for methanotrophic activity within the soil. Laboratory CH4 consumption experiments confirmed the presence of methanotrophic microorganisms in soil samples collected at Sousaki. Consumption was generally in the range from )4.9 to )38.9 pmolCH4 h )1 g but could sometimes reach extremely high values ()33 000 pmolCH4 h )1 g). These results are consistent with recent studies on other geothermal systems that revealed the existence of thermoacidophilic bacteria exerting methanotrophic activity in hot, acid soils, thereby reducing methane emissions to the atmosphere.

Molecular simulations provide new insights into the role of the accessory immunoglobulin-like domain of Cel9A

Cel9A from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius belongs to the subfamily E1 of family 9 glycoside hydrolases, many members of which have an N-terminal Ig-like domain followed by the catalytic domain. The Ig-like domain is not directly involved in either carbohydrate binding or biocatalysis; however, deletion of the Ig-domain promotes loss of enzymatic activity. We have investigated the functional role of the Ig-like domain using molecular dynamics simulations. Our simulations indicate that residues within the Ig-like domain are dynamically correlated with residues in the carbohydrate-binding pocket and with key catalytic residues of Cel9A. Free energy perturbation simulations indicate that the Ig-like domain stabilizes the catalytic domain and may be responsible for the enhanced thermostability of Cel9A.

Isolation and identification of thermo-acidophilic bacteria from orchards in china.

Eight strains of thermo-acidophilic bacteria have been isolated from apple orchards in Shaanxi Province, China. The isolated strains were identified at the species level by comparing 16S rRNA gene sequences. It was found that all strains could be assigned to two genera. The strain YL-5 belonged to Alicyclobacillus, and other isolates belonged to Bacillus. The enzymatic patterns by the API ZYM system showed very significant differences between 12 strains of Alicyclobacillus and 8 strains of Bacillus. The ability of guaiacol production varied among different strains.

Identification and characterization of a bacterial glutamic peptidase

BackgroundGlutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized.ResultsWe report the first characterization of a bacterial glutamic peptidase (pepG1), derived from the thermoacidophilic bacteria Alicyclobacillus sp. DSM 15716. The amino acid sequence identity between pepG1 and known fungal glutamic peptidases is only 24-30% but homology modeling, the presence of the glutamate/glutamine catalytic dyad and a number of highly conserved motifs strongly support the inclusion of pepG1 as a glutamic peptidase. Phylogenetic analysis places pepG1 and other putative bacterial and archaeal glutamic peptidases in a cluster separate from the fungal glutamic peptidases, indicating a divergent and independent evolution of bacterial and fungal glutamic peptidases. Purification of pepG1, heterologously expressed in Bacillus subtilis, was performed using hydrophobic interaction chromatography and ion exchange chromatography. The purified peptidase was characterized with respect to its physical properties. Temperature and pH optimums were found to be 60°C and pH 3-4, in agreement with the values observed for the fungal members of family G1. In addition, pepG1 was found to be pepstatin-insensitive, a characteristic signature of glutamic peptidases.ConclusionsBased on the obtained results, we suggest that pepG1 can be added to the MEROPS family G1 as the first characterized bacterial member.

The N-Terminal Ig Domain of Endoglucanase Cel9A from the Thermoacidophilic Alicyclobacillus Acidocaldarius Enhances Protein Stability

As part of our ongoing studies of thermophilic cellulases, we are examining the subfamily E1 of family 9 of glycoside hydrolases, members of which have an N-terminal immunoglobulin (Ig)-like domain followed by the catalytic domain (CD). While the function of the Ig-like module has not been determined, deletion of the Ig-domain results in complete loss of enzymatic activity in the cellobiohydrolase, CbhA from Clostridium thermocellum. In this work we used simulation approaches to investigate the role of the (Ig)-like domain in the non-processive endonuclease Cel9A from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius (Aa_Cel9A) for which the crystal structure has only recently been resolved.

A Novel Thermoacidophilic Cellulase from Alicyclobacillus acidocaldarius

A novel cellulase was isolated from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 grown in medium containing carboxymethylcellulose. The enzyme is a glycosylated monomer of 56.2 kDa, relatively thermostable, with optimal pH and temperature of 4.0 and 65 degrees C, respectively. Enzymatic assays on several polysaccharides demonstrated that CelG was specific for carboxymethylcellulose.

Identification of thermo-acidophilic bacteria isolated from the soil of several Japanese fruit orchards

To investigate the occurrence and distribution of thermo-acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol. Phylogenetic analysis based on the 5' end of the 16S rRNA gene (approximately 500 bp), was performed on 62 TAB isolated from the soil of several Japanese fruit orchards. The results suggested that 60 of the bacterial strains analysed belonged to the genus Alicyclobacillus, while the remaining two belonged to the genus Bacillus. The majority of strains (58%) were identified as Alicyclobacillus acidoterrestris. This group partitioned into three phylogenetically distinct subgroups (A-C). Isolates identified as A. acidiphilus (two strains), A. acidoterrestris (36 strains), and A. hesperidum subsp. aigle (one strain), produced guaiacol from vanillic acid. Levels of guaiacol production varied significantly among strains. The guaiacol producing phenotype was conserved among certain species, however no correlation was observed between levels of guaiacol production and 16S rRNA gene-based phylogenetic relatedness. Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans were widely distributed among various fruit orchards in Japan. Guaiacol production was common at the species/subspecies level; however the amount of guaiacol produced by each strain varied significantly. This study provides a comprehensive phylogenetic survey of Alicyclobacillus species in Japanese fruit orchards. Quality control standards for guaiacol producing Alicyclobacillus have also been described.

Isolation and characterization of a new family 42 β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the active site residues

The thermoacidophilic bacterium Alicyclobacillus acidocaldarius is a rich source of glycoside hydrolases enabling its growth on several di- and polysaccharides. We report here the purification and the characterization of a β-galactosidase from this source, the cloning of its gene, and the expression and the characterization of the recombinant enzyme (Aaβ-gal).The enzyme was purified 46-fold from A. acidocaldarius extracts; the gene for Aaβ-gal encoded a new member of the glycoside hydrolase family 42 (GH42) and it is flanked by a putative AraC/XylS regulator, however, the two genes were transcribed independently. The recombinant Aaβ-gal was characterized in detail revealing that it is optimally active and stable at 65 °C. Aaβ-gal is very specific for glycosides with an axial C4-OH at their non-reducing end, with k cat/ K M values of 484, 186, and 332 s − 1 mM − 1 for 2-nitrophenyl-β- d-galactoside, -fucoside, and 4-nitrophenyl-α- l-arabinoside, respectively. Finally, the characterization of the site-directed mutants Glu157Gly and Glu313Gly confirmed the latter as the nucleophile of the reaction and gave experimental evidence, for the first time in GH42, of the role of Glu157 as the acid/base of the catalyzed reaction.

고온.내산성 Bacillus sp. SJ-15를 이용한 음식물 쓰레기의 호기적 퇴비화

A thermoacidophilic bacterium was isolated from the compost and designated as Bacillus sp. SJ-15 by physiological and biochemical characteristics. The optimum temperature and pH for growth were at and pH 5.0, respectively. The strain SJ-15 was adapted in process of accelerated high-temperature composting of garbage. The highest viable cell count of composting process reached to in 16 hours. After running times of 100 days, the composting process showed a reduction rate of approximately 88%, and the concentrations of components were sufficiently high or low to satisfied the standard of organic compost except for salinity.

Effective clarification of apple juice using membrane filtration without enzyme and pasteurization pretreatment

In this study, the feasibility of clarifying apple juice using membrane filtration without enzyme-hydrolysis and pasteurization pretreatment was investigated extensively. It was found that the enzymatic and pasteurization pretreatment of apple juice had profound influence on the permeate flux and membrane fouling. The enzyme hydrolysis of the apple juice resulted in higher flux. The pasteurization of the apple juice without enzyme pretreatment caused lower flux possibly due to gelatinization at the membrane surface caused by cross-linking between deposited pectin and starch molecules. The permeate flux increased initially with the transmembrane pressure (TMP) and then kept at a plateau as TMP >2.0 bar. Generally, the permeate flux increased with the increase of cross-flow velocity (CFV). The increase of operation temperature from 23 to 50 °C also improved the permeate flux. In the long-term in situ tests without enzymatic pretreatment, the mean flux was higher than 120 l/m 2 h (LMH), and high VCF (volume concentration factor, >20) was achieved. The very stable and high flux kept over 20 hours’ industrial simulating tests also indicated that membrane fouling to the filtration performance could be neglected and the major factors influencing permeate flux were the feed concentration and viscosity. The clarified juice has very good quality in NTU, transmittance and color, and no starch, pectin and thermo-acidophilic bacteria (TAB) were detected in the clarified juice. Based on the pilot tests, a new full-scale membrane plant was designed for an apple juice producer and compared with its industrially used tubular membrane system.