Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel.

AIMS: To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces. METHODS AND RESULTS: The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy, and scanning electron microscopy (SEM) were used to evaluate the cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis, and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS). CONCLUSIONS: Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.

Cardinal models to describe the effect of temperature and pH on the growth of Anoxybacillus flavithermus & Bacillus licheniformis.

Anoxybacillus flavithermus and Bacillus licheniformis are among the predominant spore-formers of heat-processed foods. To our knowledge, no systematic analysis of growth kinetic data of A. flavithermus or B. licheniformis is currently available. In the present study, the growth kinetics of A. flavithermus and B. licheniformis in broth at various temperature and pH conditions were studied. Cardinal models were used to model the effect of the above-mentioned factors on the growth rates. The estimated values for the cardinal parameters Tmin,Topt,Tmax,pHmin and pH1/2 for A. flavithermus were 28.70 ± 0.26, 61.23 ± 0.16 and 71.52 ± 0.32 °C, 5.52 ± 0.01 and 5.73 ± 0.01, respectively, while for B. licheniformis they were 11.68 ± 0.03, 48.05 ± 0.15, 57.14 ± 0.01 °C, 4.71 ± 0.01 and 5.670 ± 0.08, respectively. The growth behaviour of these spoilers was also investigated in a pea beverage at 62 and 49 °C, respectively, to adjust the models to this product. The adjusted models were further validated at static and dynamic conditions and demonstrated good performance with 85.7 and 97.4% of predicted populations for A. flavithermus and B. licheniformis, respectively, being within the -10%-10% relative error (RE) zone. The developed models can be useful tools in assessing the potential of spoilage of heat-processed foods including plant-based milk alternatives.

Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004; A. flavithermus subsp. yunnanensis CCTCC AB2010187T Dai et al. 2011 as a later heterotypic synonym of A. tengchongensis DSM 23211T Zhang et al. 2011.

In the present study, we attempted to clarify the taxonomic positions of Anoxybacillus karvacharensis K1T, Anoxybacillus kestanbolensis NCIMB 13971T, Anoxybacillus flavithermus subsp. yunnanensis CCTCC AB2010187T, and Anoxybacillus tengchongensis DSM 23211T using whole-genome phylogenetic analysis. The genome sequence of A. kestanbolensis NCIMB13971T was not available in any database, so it was sequenced in this study. The 16S rRNA gene sequence obtained from the genome of A. kestanbolensis NCIMB13971T had 99.93% similarity with A. karvacharensis K1T. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (DDH) values between A. karvacharensis K1T and A. kestanbolensis NCIMB13971T and between A. flavithermus subsp. yunnanensis CCTCCAB 2010187T and A. tengchongensis DSM 23211T were greater than the threshold values for species demarcation. The present results indicate that A. karvacharensis K1T is a later heterotypic synonym of A. kestanbolensis NCIMB13971T; A. flavithermus subsp. yunnanensis CCTCCAB 2010187T is a later heterotypic synonym of A. tengchongensis DSM 23211T.

A new method for the preconcentrations of U(VI) and Th(IV) by magnetized thermophilic bacteria as a novel biosorbent.

This paper proposes the use of Anoxybacillus flavithermus SO-15 immobilized on iron oxide nanoparticles (NPs) as a novel magnetized biosorbent for the preconcentrations of uranium (U) and thorium (Th). The SPE procedure was based on biosorption of U(VI) and Th(IV) on a column of iron oxide NPs loaded with dead and dried thermophilic bacterial biomass prior to U(VI) and Th(IV) measurements by ICP-OES. The biosorbent characteristicswere explored using FT-IR, SEM, and EDX. Significant operational factors such as solution pH, volume and flow rate of the sample solution, amounts of dead bacteria and iron oxide nanoparticles, matrix interference effect, eluent type, and repeating use of the biosorbent on process yield were studied. The biosorption capacities were found as 62.7 and 56.4 mg g-1 for U(VI) and Th(IV), respectively. The novel extraction process has been successfullyapplied to the tap, river, and lake water samples for preconcentrations of U(VI) and Th(IV).

Resistance of thermophilic spore formers isolated from milk and whey products towards cleaning-in-place conditions: Influence of pH, temperature and milk residues.

The occurrence of thermophilic spore formers in dairy powders is a major concern for producers worldwide. This study aims to investigate the resistance of thermophilic endospores towards cleaning solutions typically used for cleaning-in-place in dairy manufacturing plants. From eleven tested strains, all were able to survive an alkaline treatment (NaOH) at 65 °C for 10 min (0.5%), whereas at concentrations of 2% eight strains withstood the treatment. Acid solutions were more sporicidal. At 0.5% of HNO3, only three strains survived the treatment. Milk impurities reduced the inactivation effect of the NaOH solutions towards thermophilic spore formers. For two selected strains, a detailed kinetic inactivation in NaOH and HNO3 solutions at different temperatures was performed and non-log-linear inactivation curves were observed. This study highlights the risk of reusing cleaning solutions in dairies.

Changes in the membrane fatty acid composition in Anoxybacillus flavithermus subsp. yunnanensis E13T as response to solvent stress.

Anoxybacillus flavithermus subsp. yunnanensis is currently the first species of strictly thermophilic bacteria that is able to tolerate a broad range of solvents. Unlike most of solvent-tolerant mesophilic bacteria, the bacterium does not synthesize unsaturated fatty acids. Our results revealed that in growing cells of A. flavithermus subsp. yunnanensis E13T, ethanol and toluene resulted in an increase in straight-chain fatty acids, mainly C16:0, leading to a more rigid membrane. Moreover, the increase in straight-chain fatty acids caused by ethanol was much higher than that of toluene. High temperature had little effect on the fatty acid composition by itself, whereas the combined conditions of high temperature and ethanol caused the dramatic increase in straight-chain fatty acids (mainly C16:0), that was balanced by decreasing branched fatty acids. The increase was also temperature dependent. The proportion of C16:0 further increased above 60 °C. No similar evidence was found in four other species of Anoxybacillus. The results suggested that A. flavithermus subsp. yunnanesis seems to develop a different response to solvents compared to its mesophilic counterparts, which consist of an increase in the saturated straight/branched ratio.

Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus.

The presence of thermophilic bacilli in dairy products is indicator of poor hygiene. Their rapid detection and identification is fundamental to improve the industrial reactivity in the implementation of corrective and preventive actions. In this study a rapid and reliable identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus was achieved by species-specific PCR assays. Two primer sets, targeting the ITS 16S-23S rRNA region and the rpoB gene sequence of the target species respectively, were employed. Species-specificity of both primer sets was evaluated by using 53 reference strains of DSMZ collection; among them, 13 species of the genus Geobacillus and 15 of the genus Anoxybacillus were represented. Moreover, 99 wild strains and 23 bulk cells collected from 24 infant formula powders gathered from several countries worldwide were included in the analyses. Both primer sets were highly specific and the expected PCR fragments were obtained only when DNA from G. stearothermophilus or A. flavithermus was used. After testing their specificity, they were combined in a Multiplex-PCR assay for the simultaneous identification of the two target species. The specificity of the Multiplex-PCR was evaluated by using both wild strains and bulk cells. Every analysis confirmed the reliable identification results provided by the single species-specific PCR methodology. The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the PCR procedures developed in this study highlight the advantage of their application for the specific detection and identification of the thermophilic species G. stearothermophilus and A. flavithermus.

A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of Anoxybacillus flavithermus.

Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, Anoxybacillus flavithermus, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn2+ ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs.

Isolation and characterization of potential multiple extracellular enzyme-producing bacteria from waste dumping area in Addis Ababa.

Extremozymes are innovative and robust biocatalysts produced by various microorganisms from harsh environments. As thermophilic organisms can only develop in a few places, studying them in geothermal environments has provided new insights into the origins and evolution of early life and access to significant bio-resources with potential biotechnology applications. The work aimed to isolate and identify likely multiple extracellular enzyme-producing thermophilic bacteria from an Addis Ababa landfill (Qoshe). The streaking approach was used to purify 102 isolates acquired by serial dilution and spread plate method. The isolates were morphologically and biochemically characterized. Thirty-five cellulases, 22 amylase, 17 protease, and nine lipase-producing bacteria were identified using primary screening methods. Further secondary screening using Strain safety evaluation; two bacterial strains (TQ11 and TQ46) were identified. Based on morphological and biochemical tests, they were found to be gram-positive and rod-shaped. Furthermore, molecular identification and phylogenic analysis of selected promising isolates confirmed the identity of the isolates, Paenibacillus dendritiformis (TQ11) and Anoxybacillus flavithermus (TQ46). The results indicated that, multiple extracellular enzyme-producing thermophilic bacteria isolated from a waste dumping area in Addis Ababa offer useful features for environmental sustainability in a wide range of industrial applications due to their biodegradability and specialized stability under extreme conditions, increased raw material utilization, and decreased waste.

Exploration on the Cr(VI) resistance mechanism of a novel thermophilic Cr(VI)-reducing bacteria Anoxybacillus flavithermus ABF1 isolated from Tengchong geothermal region, China.

Hexavalent chromium resistance and reduction mechanisms of microorganism provide a critical guidance for Cr(VI) bioremediation. However, related researches are limited in mesophiles and deficient for thermophiles. In this work, a novel alkaline Cr(VI)-reducing thermophile Anoxybacillus flavithermus ABF1 was isolated from geothermal region. The mechanisms of Cr(VI) resistance and reduction were investigated. The results demonstrated that A. flavithermus ABF1 could survive in a wide temperature range from 50°C to 70°C and in pH range of 7.0-9.0. Strain ABF1 showed excellent growth activity and Cr(VI) removal performance when initial Cr(VI) concentration was lower than 200 mg L-1 . 93.71% of Cr(VI) was removed at initial concentration of 20 mg L-1 after 72 h. The majority of Cr(VI) was found to be reduced extracellularly by enzymes secreted by cells. XPS and Raman analysis further manifested that Cr2 O3 was the product of Cr(VI) reduction. Moreover, the Cr(VI) transportation-related gene cysP and Cr(VI) reduction-related gene azoR of A. flavithermus ABF1 played key roles in inhibiting Cr(VI) entering cells and promoting extracellular Cr(VI) reduction respectively. This work provides novel insight into the mechanisms of Cr(VI) resistance and detoxication of thermophiles, which leads to a promising alternative strategy for heavy metal bioremediation in areas with elevated temperature.

Immunostimulatory activities of specific bacterial secondary metabolite of Anoxybacillus flavithermus strain SX-4 on carp, Cyprinus carpio.

AIMS: To determine the capacity of secondary metabolite of strain SX-4, to enhance the nonspecific immunity and survival of carp (Cyprinus carpio), and to identify the constituents that are responsible. METHODS AND RESULTS: A thermophilic strain SX-4 that is able to produce immunostimulatory metabolite was isolated from sludge sample of hot spring and identified by comparison with 16S rRNA sequences (99% of homology) as Anoxybacillus flavithermus. Bioactivity-guided fractionation of methanol extract from its cell-free culture, one bacterial peptide with the capacity of improving the nonspecific immune responses and disease resistance (relative per cent survival = 66·67%) was obtained and the compound was characterized as cyclo-(L-Pro-Gly) by IR, ESI-MS, (1) H NMR and (13) C NMR spectroscopic analyses. After intraperitoneal administration of this peptide, selected innate immune parameters including phagocytic activity, superoxide anion production, serum lysozyme activity and serum SOD activity, along with immune-related genes expression (i.e. interleukin-1ß and inducible nitric oxide synthase), in the blood were found to be significantly increased. CONCLUSIONS: The bacterial peptide cyclo-(L-Pro-Gly) significantly enhances nonspecific immunity and survival of carp. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a possibility of using cyclo-(L-Pro-Gly) as a better natural immunostimulant, which could have a promising role in aquaculture to prevent diseases and disease outbreaks.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

Thermal treatment of skim milk concentrates in a novel shear-heating device: Reduction of thermophilic spores and physical properties.

Endospores of thermophilic bacilli are a major concern for producers of dairy powders. In this study, we heat treated 10 different spore suspensions at 110 °C in skim milk and skim milk concentrate (36% dry matter) of the species Geobacillus stearothermophilus (10 min) and Anoxybacillus flavithermus (5 min) in a new shear-heating device. The highest log reduction in skim milk concentrate was 3.5. The death behavior of the spores was strain dependent. Particle formation and Maillard reaction were observed. By increasing the shear-rate up to 1500 s-1 the particle size was reduced for both heating times (D90 reduction: 57.4 and 77.0%, respectively). The particle size was lessened by a reduction of dry matter of 27%, compared to 36%. This work emphasizes, that heat treatment of concentrated dairy products represents a technological option to reduce thermophilic spores in skim milk concentrate and powders produced thereof.

Draft genome sequence of Anoxybacillus flavithermus KU2-6-11 isolated from hot-spring in Uzon caldera (Kamchatka, Russia).

The Anoxybacillus flavithermus KU2-6-11 was isolated from sediments of a nameless hot spring. The hot spring is located in Uzon caldera (Kamchatka, Russia). The sequenced and annotated genome is 2,646,305 bp and encodes 2787genes. The draft genome sequence of the Anoxybacillus flavithermus KU2-6-11 has been deposited at DDBJ/EMBL/GenBank under the accession PEDM01000000 and the sequences could be found at the site https://www.ncbi.nlm.nih.gov/nuccore/PEDM01000000.

Purification and characterization of thermostable α-amylase from thermophilic Anoxybacillus flavithermus.

This study reports on the purification and characterization of thermostable α-amylase (α-1-4 D-glucan glucanohydrolase EC 3.2.1.1) from a newly isolated Anoxybacillus flavithermus. A. flavithermus was used, which was isolated from hot water springs of Ömer, Afyonkarahisar, Turkey. The gram-positive, spore-forming, motile, moderately thermophilic bacteria were found to be a strain of A. flavithermus analysed by 16S rRNA comparison. The optimal conditions for bacterial growth were determined to be at 20 thh, 55 °C and pH 6.0. Maximum α-amylase activity was obtained at 55 °C at pH 7.0 after 24h of incubation. Thermostable α-amylase from A. flavithermus was purified by 70% (NH4)2SO4 and ion-exchange chromatography (5.2-fold; 65.8% yield). The molecular weight of α-amylase was 60 kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The α-amylase hydrolyzed soluble starch at 55 °C with Km: 0.005 mM and Vmax: 3.5 µmol min(-1).

Non-contiguous finished genome sequence of Anoxybacillus flavithermus subsp. yunnanensis type strain (E13(T)), a strictly thermophilic and organic solvent-tolerant bacterium.

Anoxybacillus flavithermus subsp. yunnanensis is the only strictly thermophilic bacterium that is able to tolerate a broad range of toxic solvents at its optimal temperature of 55-60°C. The type strain E13(T) was isolated from water-sediment slurries collected from a hot spring. This study presents the draft genome sequence of A. flavithermus subsp. yunnanensis E13(T) and its annotation. The 2,838,393bp long genome (67 contigs) contains 3,035 protein-coding genes and 85 RNA genes, including 10 rRNA genes, and no plasmids. The genome information has been used to compare with the genomes from A. flavithermus subsp. flavithermus strains.

Novel physico-biological treatment for the remediation of textile dyes-containing industrial effluents.

In this work, a novel remediation strategy consisting of a sequential biological and physical process is proposed to remove dyes from a textile polluted effluent. The decolorization ability of Anoxybacillus flavithermus in an aqueous effluent containing two representative textile finishing dyes (Reactive Black 5 and Acid Black 48, as di-azo and antraquinone class, respectively) was proved. The decolorization efficiency for a mixture of both dyes reached almost 60% in less than 12h, which points out the suitability of the selected microorganism. In a sequential stage, an aqueous biphasic system consisting of non-ionic surfactants and a potassium-based organic salt, acting as the salting out agent, was investigated. The phase segregation potential of the selected salts was evaluated in the light of different thermodynamic models, and remediation levels higher than 99% were reached.