Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.

Exploration of isoxanthohumol bioconversion from spent hops into 8-prenylnaringenin using resting cells of Eubacterium limosum.

Hops is an almost unique source of the potent phytoestrogen 8-prenylnaringenin (8-PN). As hops contain only low levels of 8-PN, synthesis may be more attractive than extraction. A strain of the Gram-positive Eubacterium limosum was isolated previously for 8-PN production from more abundant precursor isoxanthohumol (IX) from hops. In this study, spent hops, an industrial side stream from the beer industry, was identified as interesting source of IX. Yet, hop-derived compounds are well-known antibacterial agents and the traces of a large variety of different compounds in spent hops interfered with growth and IX conversion. Critical factors to finally enable bacterial 8-PN production from spent hops, using a food and feed grade medium, were evaluated in this research. The use of bacterial resting cells and complex medium at a pH of 7.8-8 best fulfilled the requirements for 8-PN production and generated a solid basis for development of an economic process.

Single Nucleotide Polymorphisms and Biochemical Markers As Complementary Tools To Characterize Hops ( Humulus lupulus L.) in Brewing Practice.

In brewing practice, the use of the appropriate hop variety is essential to produce consistent and high-quality beers. Yet, hop batches of the same variety cultivated in different geographical regions can display significant biochemical differences, resulting in specific taste- and aroma-related characteristics in beer. In this study, we illustrate the complementarity of genetic and biochemical fingerprinting methods to fully characterize hop batches. Using genotyping-by-sequencing (GBS), a set of 1 830 polymorphic single nucleotide polymorphism (SNP) markers generated 48 unique genetic fingerprints for a collection of 56 commercial hop varieties. Three groups of varieties, consisting of somaclonal variants, could not be further differentiated using this set of markers. Biochemical marker information offered added value to characterize hop samples from a given variety grown at different geographical locations. We demonstrate the power of combining genetic and biochemical fingerprints for quality control of hop batches in the brewing industry.

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted ß-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

Ultra-High-Performance Supercritical Fluid Chromatography as a Separation Tool for Fusarium Mycotoxins and Their Modified Forms.

A simple, reliable method for the detection of free and modified Fusarium mycotoxins in beer using state-of-the-art ultra-high-performance supercritical fluid chromatography (UHPSFC) with low-resolution tandem MS (MS/MS) is presented in this paper. The UHPSFC-MS/MS method was developed for nivalenol, deoxynivalenol, 15-acetyl-deoxynivalenol, 3-acetyl-deoxynivalenol, deoxynivalenol-3-glucoside, HT-2 toxin, T-2 toxin, T-2 toxin-3-glucoside, neosolaniol, diacetoxyscirpenol, zearalenone, α-zearalenol, and ß-zearalenol and their internal standards deepoxy-deoxynivalenol and zearalanone. Due to the broad range of the physicochemical properties of the aforementioned, the sample preparation step was minimized to avoid analyte losses. Extraction with acetonitrile-water-acetic acid (79 + 20 + 1, v/v/v) and hexane in combination with solid-phase extraction (C18) was followed by a filtration step. After filtration, the extract was evaporated, and the remaining residue was redissolved in a mobile phase for injection (methanol-water; 90 + 10, v/v). A mobile phase consisting of supercritical CO2 and a small portion of methanol was used. The developed multimycotoxin method permits the simultaneous determination of multiple fusariotoxins in an one-step chromatographic run using UHPSFC-MS/MS. SFC is a promising strategy; however, the retention mechanism is complex, leading to the unpredictable nature of elution and to some mycotoxins not being retained on the column. This restricts the applicability of UHPSFC in multimycotoxin analyses. The present study is the first report on the use of UHPSFC for the analysis of free and modified Fusarium mycotoxins.

Plant-Based Beverages as Good Sources of Free and Glycosidic Plant Sterols.

To address the ever-growing group of health-conscious consumers, more and more nutritional and health claims are being used on food products. Nevertheless, only very few food constituents, including plant sterols, have been appointed an approved health claim (European Commission and Food and Drugs Administration). Plant sterols are part of those limited lists of approved compounds for their cholesterol-lowering properties but have been praised for their anti-inflammatory and anti-carcinogenic properties as well. Despite this indisputable reputation, direct quantitative data is still lacking for naturally present (conjugated) plant sterols in beverages. This study aimed to fill this gap by applying a validated extraction and UPLC-MS/MS detection method to a diverse range of everyday plant-based beverages. ß-sitosterol-ß-d-glucoside (BSSG) showed to be by far the most abundant sterol in all beverages studied, with concentrations up to 60-90 mg per 100 mL in plant-based milk alternatives and fresh fruit juices. Ergosterol (provitamin D2) could be found in beers (0.8-6.1 µg per 100 mL, from the yeast) and occasionally in juices (17-29 µg per 100 mL). Overall, the results demonstrated that the concentrations of water-soluble sterol conjugates have been underestimated significantly and that specific plant-based beverages can be good, low-fat sources of these plant sterols.

Fractional factorial design-based optimisation and application of an extraction and UPLC-MS/MS detection method for the quantification of phytosterols in food, feed and beverages low in phytosterols.

Phytosterols are ubiquitous in plants, as they play an important role in cell membrane stability and as signal transducers. Over the last few decades, scientific interest in phytosterols has significantly increased. Most of the interest has focused on the cholesterol-lowering properties of phytosterols, but they may also interfere with endogenous steroid hormone synthesis. Despite this dual interest in phytosterols, accurate and fully validated methods for the quantification of phytosterols in food and feed samples are scarce. During this study an extraction and detection method for the main free phytosterols (ß-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria, reaching all evaluated performance parameter requirements. The individual recoveries ranged between 95 and 104 %. Good results for repeatability and intralaboratory reproducibility (RSD %) were observed (<10 %). Excellent linearity was proven on the basis of determination coefficient (R 2 > 0.99) and lack-of-fit test (F test, alpha = 0.05). The limits of detection (LODs) and lower limits of quantification (LLOQs) in grain matrices were as low as 0.01-0.03 mg per 100 g and 0.02-0.10 mg per 100 g. This method allowed quantification of all main, free phytosterols in different grains (oats, barley, corn, malt) and it was shown that the method can be used for other solid food and feed samples as well, including new matrices such as straw, hay, mustard seeds, grass and yellow peas. Additionally, the method was shown to perform well in liquid samples low in phytosterols such as concentrate-based juices, soft drinks and beers (<5 µg per 100 mL). Graphical Abstract An extraction and detection method for the main free phytosterols (ß-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria and applied on different matrices including feed and beverages.

Validated UPLC-MS/MS Methods To Quantitate Free and Conjugated Alternaria Toxins in Commercially Available Tomato Products and Fruit and Vegetable Juices in Belgium.

Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 µg/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (

Microbial diversity and metabolite composition of Belgian red-brown acidic ales.

Belgian red-brown acidic ales are sour and alcoholic fermented beers, which are produced by mixed-culture fermentation and blending. The brews are aged in oak barrels for about two years, after which mature beer is blended with young, non-aged beer to obtain the end-products. The present study evaluated the microbial community diversity of Belgian red-brown acidic ales at the end of the maturation phase of three subsequent brews of three different breweries. The microbial diversity was compared with the metabolite composition of the brews at the end of the maturation phase. Therefore, mature brew samples were subjected to 454 pyrosequencing of the 16S rRNA gene (bacteria) and the internal transcribed spacer region (yeasts) and a broad range of metabolites was quantified. The most important microbial species present in the Belgian red-brown acidic ales investigated were Pediococcus damnosus, Dekkera bruxellensis, and Acetobacter pasteurianus. In addition, this culture-independent analysis revealed operational taxonomic units that were assigned to an unclassified fungal community member, Candida, and Lactobacillus. The main metabolites present in the brew samples were L-lactic acid, D-lactic acid, and ethanol, whereas acetic acid was produced in lower quantities. The most prevailing aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, which might be of impact on the aroma of the end-products.

The microbial diversity of an industrially produced lambic beer shares members of a traditionally produced one and reveals a core microbiota for lambic beer fermentation.

The microbiota involved in lambic beer fermentations in an industrial brewery in West-Flanders, Belgium, was determined through culture-dependent and culture-independent techniques. More than 1300 bacterial and yeast isolates from 13 samples collected during a one-year fermentation process were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by sequence analysis of rRNA and various protein-encoding genes. The bacterial and yeast communities of the same samples were further analyzed using denaturing gradient gel electrophoresis of PCR-amplified V3 regions of the 16S rRNA genes and D1/D2 regions of the 26S rRNA genes, respectively. In contrast to traditional lambic beer fermentations, there was no Enterobacteriaceae phase and a larger variety of acetic acid bacteria were found in industrial lambic beer fermentations. Like in traditional lambic beer fermentations, Saccharomyces cerevisiae, Saccharomyces pastorianus, Dekkera bruxellensis and Pediococcus damnosus were the microorganisms responsible for the main fermentation and maturation phases. These microorganisms originated most probably from the wood of the casks and were considered as the core microbiota of lambic beer fermentations.

Microbiota and metabolites of aged bottled gueuze beers converge to the same composition.

Gueuze beers are prepared by mixing young and old lambic beers and are bottle-refermented spontaneously for aging. The present study analyzed the microbiota and metabolites present in gueuze beers that were aged between a few months and up to 17 years. Yeasts were cultivated from all beers sampled, but bacteria could not be grown from beers older than 5 years. Lactic acid and ethyl lactate concentrations increased steadily during aging, whereas ethanol concentrations remained constant. The concentrations of isoamyl acetate and ethyl decanoate decreased during the aging process. Hence, ethyl lactate and ethyl decanoate can be considered as positive and negative gueuze beer-aging metabolite biomarkers, respectively. Nevertheless, considerable bottle-to-bottle variation in the metabolite profiles was found, which hindered the generalization of the effects seen during the aging of the gueuze beers examined, but which illustrated the unique character of the lambic beers. The present results further indicate that gueuze beers are preferably aged for less than 10 years.

Development and validation of an ultra-high-performance liquid chromatography tandem mass spectrometric method for the simultaneous determination of free and conjugated Alternaria toxins in cereal-based foodstuffs.

A UPLC-ESI+/--MS/MS method for the simultaneous determination of free (alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid, tentoxin, altertoxin-I) and conjugated (sulfates and glucosides of alternariol and alternariol monomethyl ether) Alternaria toxins in cereals and cereal products (rice, oat flakes and barley) was developed. Optimization of the sample preparation and extraction methodology was achieved through experimental design, using full factorial design for extraction solvent composition optimization and fractional factorial design to identify the critical factors in the sample preparation protocol, which were in turn subjected to optimization. Final extracts were analysed using an Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer equipped with an electrospray interface operated in both positive and negative ionization mode. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column, and the applied gradient elution programme allowed for the simultaneous determination of 10 Alternaria toxins in a one-step chromatographic run with a total run time of only 7min. Subsequently, the method, applying isotopically labelled internal standards ([2H4]-alternariol monomethyl ether and [13C6,15N]-tenuazonic acid), was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, measurement uncertainty and specificity (in agreement with the criteria mentioned in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC). During validation, quality of the bioanalytical data was improved by counteracting the observed heteroscedasticity through the application of weighted least squares linear regression (WLSLR). Finally, 24 commercially available cereal-based foodstuffs were subjected to analysis, revealing the presence of tenuazonic acid in both rice and oat flake samples (

Identification of beer-spoilage bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of beer-spoilage bacteria was examined. To achieve this, an extensive identification database was constructed comprising more than 4200 mass spectra, including biological and technical replicates derived from 273 acetic acid bacteria (AAB) and lactic acid bacteria (LAB), covering a total of 52 species, grown on at least three growth media. Sequence analysis of protein coding genes was used to verify aberrant MALDI-TOF MS identification results and confirmed the earlier misidentification of 34 AAB and LAB strains. In total, 348 isolates were collected from culture media inoculated with 14 spoiled beer and brewery samples. Peak-based numerical analysis of MALDI-TOF MS spectra allowed a straightforward species identification of 327 (94.0%) isolates. The remaining isolates clustered separately and were assigned through sequence analysis of protein coding genes either to species not known as beer-spoilage bacteria, and thus not present in the database, or to novel AAB species. An alternative, classifier-based approach for the identification of spoilage bacteria was evaluated by combining the identification results obtained through peak-based cluster analysis and sequence analysis of protein coding genes as a standard. In total, 263 out of 348 isolates (75.6%) were correctly identified at species level and 24 isolates (6.9%) were misidentified. In addition, the identification results of 50 isolates (14.4%) were considered unreliable, and 11 isolates (3.2%) could not be identified. The present study demonstrated that MALDI-TOF MS is well-suited for the rapid, high-throughput and accurate identification of bacteria isolated from spoiled beer and brewery samples, which makes the technique appropriate for routine microbial quality control in the brewing industry.

The microbial diversity of traditional spontaneously fermented lambic beer.

Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

Acetobacter sicerae sp. nov., isolated from cider and kefir, and identification of species of the genus Acetobacter by dnaK, groEL and rpoB sequence analysis.

Five acetic acid bacteria isolates, awK9_3, awK9_4 ( = LMG 27543), awK9_5 ( = LMG 28092), awK9_6 and awK9_9, obtained during a study of micro-organisms present in traditionally produced kefir, were grouped on the basis of their MALDI-TOF MS profile with LMG 1530 and LMG 1531(T), two strains currently classified as members of the genus Acetobacter. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences as well as on concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB indicated that these isolates were representatives of a single novel species together with LMG 1530 and LMG 1531(T) in the genus Acetobacter, with Acetobacter aceti, Acetobacter nitrogenifigens, Acetobacter oeni and Acetobacter estunensis as nearest phylogenetic neighbours. Pairwise similarity of 16S rRNA gene sequences between LMG 1531(T) and the type strains of the above-mentioned species were 99.7%, 99.1%, 98.4% and 98.2%, respectively. DNA-DNA hybridizations confirmed that status, while amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) data indicated that LMG 1531(T), LMG 1530, LMG 27543 and LMG 28092 represent at least two different strains of the novel species. The major fatty acid of LMG 1531(T) and LMG 27543 was C18 : 1ω7c. The major ubiquinone present was Q-9 and the DNA G+C contents of LMG 1531(T) and LMG 27543 were 58.3 and 56.7 mol%, respectively. The strains were able to grow on D-fructose and D-sorbitol as a single carbon source. They were also able to grow on yeast extract with 30% D-glucose and on standard medium with pH 3.6 or containing 1% NaCl. They had a weak ability to produce acid from d-arabinose. These features enabled their differentiation from their nearest phylogenetic neighbours. The name Acetobacter sicerae sp. nov. is proposed with LMG 1531(T) ( = NCIMB 8941(T)) as the type strain.

Gluconobacter cerevisiae sp. nov., isolated from the brewery environment.

Three strains, LMG 27748(T), LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748(T) revealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA-DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39-65 %). The three strains used d-fructose, d-sorbitol, meso-erythritol, glycerol, l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748(T) and LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 °C. They produced acid from meso-erythritol and sucrose, but not from raffinose. d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced from d-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748(T), LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species, Gluconobacter cerevisiae sp. nov., with LMG 27748(T) ( = DSM 27644(T)) as the type strain.

Acetobacter lambici sp. nov., isolated from fermenting lambic beer.

An acetic acid bacterium, strain LMG 27439(T), was isolated from fermenting lambic beer. The cells were Gram-stain-negative, motile rods, catalase-positive and oxidase-negative. Analysis of the 16S rRNA gene sequence revealed the strain was closely related to Acetobacter okinawensis (99.7 % 16S rRNA gene sequence similarity with the type strain of this species), A. ghanensis (99.6 %), A. syzygii (99.6 %), A. fabarum (99.4 %) and A. lovaniensis (99.2 %). DNA-DNA hybridization with the type strains of these species revealed moderate DNA-DNA hybridization values (31-45 %). Strain LMG 27439(T) was unable to grow on glycerol or methanol as the sole carbon source, on yeast extract with 10 % ethanol or on glucose-yeast extract medium at 37 °C. It did not produce acid from l-arabinose, d-galactose or d-mannose, nor did it produce 2-keto-d-gluconic acid, 5-keto-d-gluconic acid or 2,5-diketo-d-gluconic acid from d-glucose. It did not grow on ammonium as the sole nitrogen source and ethanol as the sole carbon source. These genotypic and phenotypic data distinguished strain LMG 27439(T) from established species of the genus Acetobacter, and therefore we propose this strain represents a novel species of the genus Acetobacter. The name Acetobacter lambici sp. nov. is proposed, with LMG 27439(T) ( = DSM 27328(T)) as the type strain.

Effects of growth medium on matrix-assisted laser desorption-ionization time of flight mass spectra: a case study of acetic acid bacteria.

The effect of the growth medium used on the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determined by using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 mass spectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound effect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when culture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation but strongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF mass spectrometry identification database should comprise mass spectra of multiple reference strains per species grown on different culture media to facilitate species and strain level differentiation.

An rpoD gene sequence based evaluation of cultured Pseudomonas diversity on different growth media.

The last decade has shown an increased interest in the utilization of bacteria for applications ranging from bioremediation to wastewater purification and promotion of plant growth. In order to extend the current number of micro-organism mediated applications, a continued quest for new agents is required. This study focused on the genus Pseudomonas, which is known to harbour strains with a very diverse set of interesting properties. The aim was to identify growth media that allow retrieval of a high Pseudomonas diversity, as such increasing the chance of isolating isolates with beneficial properties. Three cultivation media: trypticase soy agar (TSA), potato dextrose agar (PDA) and Pseudomonas isolation agar (PIA) were evaluated for their abilities to grow Pseudomonas strains. TSA and PDA were found to generate the largest Pseudomonas diversity. However, communities obtained with both media overlapped. Communities obtained with PIA, on the other hand, were unique. This indicated that the largest diversity is obtained by sampling from either PDA or TSA and from PIA in parallel. To evaluate biodiversity of the isolated Pseudomonas members on the media, an appropriate biomarker had to be identified. Hence, an introductory investigation of the taxonomic resolution of the 16S rRNA, rpoD, gyrB and rpoB genes was performed. The rpoD gene sequences not only had a high phylogenetic content and the highest taxonomic resolution amongst the genes investigated, it also had a gene phylogeny that related well with that of the 16S rRNA gene.

Hazenella coriacea gen. nov., sp. nov., isolated from clinical specimens.

A Gram-staining-positive, endospore-forming rod was isolated independently from clinical specimens in New York State, USA, once in 2009 and twice in 2011. The three isolates had identical 16S rRNA gene sequences and, based on their 16S rRNA gene sequence, are most closely related to the type strains of Laceyella sediminis and L. sacchari (94.6 % similarity). The partial 23S rRNA gene sequences of the three strains were also 100 % identical. Maximum-likelihood phylogenetic analysis suggests that the new isolates belong to the family Thermoactinomycetaceae. Additional biochemical and phenotypic characteristics of the strains support the family designation and suggest that the three isolates represent a single species. In each of the strains, the predominant menaquinone is MK-7, the diagnostic diamino acid is meso-diaminopimelic acid and the major cellular fatty acids are iso-C15 : 0, anteiso-C15 : 0 and iso-C13 : 0. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, four unknown phospholipids, four unknown aminophospholipids and an unknown lipid. It is proposed that the novel isolates represent a single novel species within a new genus, for which the name Hazenella coriacea gen. nov., sp. nov. is proposed. The type strain of Hazenella coriacea is strain 23436(T) ( = DSM 45707(T) = LMG 27204(T)).