Development of SARS-CoV-2 packaged RNA reference material for nucleic acid testing.

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.

Nucleic Acid Testing of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.

Kitasatospora acidiphila sp. nov., isolated from pine grove soil, exhibiting antimicrobial potential.

A polyphasic study was carried out to establish the taxonomic position of an acidophilic isolate designated MMS16-CNU292T (=JCM 32302T) from pine grove soil, and provisionally assigned to the genus Kitasatospora. On the basis of 16S rRNA gene sequence similarity, the strain formed a novel evolutionary lineage within Kitasatospora and showed highest similarities to Kitasatospora azatica KCTC 9699T (98.75 %), Kitasatospora kifunensis IFO 15206T (98.74 %), Kitasatospora purpeofusca NRRL B-1817T (98.61 %) and Kitasatospora nipponensis HKI 0315T (98.42 %), respectively. Strain MMS16-CNU292T possessed MK-9(H6) and MK-9(H8) as the major menaquinones, and a major amount of meso-diaminopimelic acid in the cell-wall peptidoglycan. The whole-cell hydrolysates were rich in galactose, glucose and mannose, and the polar lipids mainly consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannosides. The major fatty acids were anteiso-C15 : 1-A, anteiso-C15 : 0, and iso-C15 : 0, and the DNA G+C content was 71.5 mol%. The strain exhibited antibacterial activity against a number of bacterial strains, and the activity was generally greater when grown in acidic conditions. The phylogenetic, chemotaxonomic and phenotypic properties enabled distinction of MMS16-CNU292T from related species, and thus the isolate should be recognized as a new species of the genus Kitasatospora, for which the name Kitasatospora acidiphila sp. nov. (type strain=MMS16-CNU292T=KCTC 49011T=JCM 32302T) is proposed.

Herpes Simplex Virus 1 Specifically Targets Human CD1d Antigen Presentation To Enhance Its Pathogenicity.

Herpes simplex virus 1 (HSV-1) is one of the most prevalent herpesviruses in humans and represents a constant health threat to aged and immunocompromised populations. How HSV-1 interacts with the host immune system to efficiently establish infection and latency is only partially known. CD1d-restricted NKT cells are a critical arm of the host innate immune system and play potent roles in anti-infection and antitumor immune responses. We discovered previously that upon infection, HSV-1 rapidly and efficiently downregulates CD1d expression on the cell surface and suppresses the function of NKT cells. Furthermore, we identified the viral serine/threonine protein kinase US3 as a major viral factor downregulating CD1d during infection. Interestingly, neither HSV-1 nor its US3 protein efficiently inhibits mouse CD1d expression, suggesting that HSV-1 has coevolved with the human immune system to specifically suppress human CD1d (hCD1d) and NKT cell function for its pathogenesis. This is consistent with the fact that wild-type mice are mostly resistant to HSV-1 infection. On the other hand, in vivo infection of CD1d-humanized mice (hCD1d knock-in mice) showed that HSV-1 can indeed evade hCD1d function and establish infection in these mice. We also report here that US3-deficient viruses cannot efficiently infect hCD1d knock-in mice but infect mice lacking all NKT cells at a higher efficiency. Together, these studies supported HSV-1 evasion of human CD1d and NKT cell function as an important pathogenic factor for the virus. Our results also validated the potent roles of NKT cells in antiherpesvirus immune responses and pointed to the potential of NKT cell ligands as adjuvants for future vaccine development.IMPORTANCE Herpes simplex virus 1 (HSV-1) is among the most common human pathogens. Little is known regarding the exact mechanism by which this virus evades the human immune system, particularly the innate immune system. We reported previously that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule, CD1d, so as to evade the antiviral function of NKT cells. Here we demonstrated that the virus has coevolved with the human CD1d and NKT cell system and that NKT cells indeed play potent roles in anti-HSV immune responses. These studies point to the great potential of exploring NKT cell ligands as adjuvants for HSV vaccines.

Complete Genome Sequence of Paenibacillus sp. CAA11: A Promising Microbial Host for Lignocellulosic Biorefinery with Consolidated Processing.

Several bioprocessing technologies, such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP), have been highlighted to produce bio-based fuels and chemicals from lignocellulosic biomass. Successful CBP, an efficient and economical lignocellulosic biorefinery process compared with other processes, requires microorganisms with sufficient cellulolytic activity and biofuel/chemical-producing ability. Here, we report the complete genome of Paenibacillus sp. CAA11, a newly isolated promising microbial host for CBP-producing ethanol and organic acids from cellulose. The genome of Paenibacillus sp. CAA11 comprises one 4,888,410 bp chromosome with a G + C content of 48.68% containing 4418 protein-coding genes, 102 tRNA genes, and 39 rRNA genes. The functionally active cellulase, encoded by CAA_GH5 was identified to belong to glycosyl hydrolase family 5 (GH5) and consisted of a catalytic domain and a cellulose-binding domain 3 (CBM3). When cellulolytic activity of CAA_GH5 was assayed through Congo red method by measuring the size of halo zone, the recombinant Bacillus subtilis RIK1285 expressing CAA_GH5 showed a comparable cellulolytic activity to B. subtilis RIK1285 expressing Cel5, a previously verified powerful bacterial cellulase. This study demonstrates the potential of Paenibacillus sp. CAA11 as a CBP-enabling microbe for cost-effective biofuels/chemicals production from lignocellulosic biomass.

Megasphaera hexanoica sp. nov., a medium-chain carboxylic acid-producing bacterium isolated from a cow rumen.

Strain MHT, a strictly anaerobic, Gram-stain-negative, non-spore-forming, spherical coccus or coccoid-shaped microorganism, was isolated from a cow rumen during a screen for hexanoic acid-producing bacteria. The microorganism grew at 30-40 °C and pH 5.5-7.5 and exhibited production of various short- and medium-chain carboxylic acids (acetic acid, butyric acid, pentanoic acid, isobutyric acid, isovaleric acid, hexanoic acid, heptanoic acid and octanoic acid), as well as H2 and CO2 as biogas. Phylogenetic analysis based on 16S rRNA gene sequencing demonstrated that MHT represents a member of the genus Megasphaera, with the closest relatives being Megapsphaera indica NMBHI-10T (94.1 % 16S rRNA sequence similarity), Megasphaera elsdenii DSM 20460T (93.8 %) and Megasphaera paucivorans DSM 16981T (93.8 %). The major cellular fatty acids produced by MHT included C12 : 0, C16 : 0, C18 : 1cis 9, and C18 : 0, and the DNA G+C content of the MHT genome is 51.8 mol%. Together, the distinctive phenotypic and phylogenetic characteristics of MHT indicate that this microorganism represents a novel species of the genus Megasphaera, for which the name Megasphaera hexanoica sp. nov. is herein proposed. The type strain of this species is MHT (=KCCM 43214T=JCM 31403T).

A Subset of CD8αß+ Invariant NKT Cells in a Humanized Mouse Model.

Invariant NKT (iNKT) cells are unconventional innate-like T cells demonstrating potent antitumor function in conventional mouse models. However, the iNKT cell ligands have had limited efficacy in human antitumor clinical trials, mostly due to the profound differences in the properties and compositions of iNKT cells between the two species, including the presence of a CD8(+) subset of iNKT cells only in humans. To build reliable in vivo models for studying human iNKT cells, we recently developed the first humanized mouse model (hCD1d-KI) with human CD1d knocked in. To further humanize the mouse model, we now introduced the human invariant NKT TCRα-chain (Vα24Jα18) into the hCD1d-knockin mice. Similar to humans, this humanized mouse model developed a subset of CD8αß(+) iNKT cells among other human-like iNKT subsets. The presence of the CD8αß(+) iNKT cells in the thymus suggests that these cells developed in the thymus. In the periphery, these NKT cells showed a strong Th1-biased cytokine response and potent cytotoxicity for syngeneic tumor cells upon activation, as do human CD8αß(+) iNKT cells. The low binding avidity of iNKT TCRs to the human CD1d/lipid complex and high prevalence of Vß7 TCRß among the CD8(+) iNKT cells strongly point to a low avidity-based developmental program for these iNKT cells, which included the suppression of Th-POK and upregulation of eomesodermin transcriptional factors. Our establishment of this extensively humanized mouse model phenotypically and functionally reflecting the human CD1d/iNKT TCR system will greatly facilitate the future design and optimization of iNKT cell-based immunotherapies.

Herpes Simplex Virus 1 US3 Phosphorylates Cellular KIF3A To Downregulate CD1d Expression.

UNLABELLED: Herpes simplex virus 1 (HSV-1) causes one of the most prevalent herpesviral infections in humans and is the leading etiological agent of viral encephalitis and eye infections. Our understanding of how HSV-1 interacts with the host at the cellular and organismal levels is still limited. We and others previously reported that, upon infection, HSV-1 rapidly and efficiently downregulates CD1d cell surface expression and suppresses the function of NKT cells, a group of innate T cells with critical immunoregulatory function. The viral protein kinase US3 plays a major role in this immune evasion mechanism, and its kinase activity is required for this function. In this study, we investigated the cellular substrate(s) phosphorylated by US3 and how it mediates US3 suppression of CD1d recycling. We identified the type II kinesin motor protein KIF3A as a critical kinesin factor in the cell surface expression of CD1d. Interestingly, KIF3A is phosphorylated by US3 both in vitro and in infected cells. Mass spectrometry analysis of purified KIF3A showed that it is phosphorylated predominantly at serine 687 by US3. Ablation of this phosphorylation abolished US3-mediated downregulation of CD1d expression, suggesting that phosphorylation of KIF3A is the primary mechanism of HSV-1 suppression of CD1d expression by US3 protein. Understanding of the precise mechanism of viral modulation of CD1d expression will help to develop more efficient vaccines in the future to boost host NKT cell-mediated immune responses against herpesviruses. IMPORTANCE: Herpes simplex virus 1 (HSV-1) is among the most common human pathogens. Little is known regarding the exact mechanism by which this virus evades the human immune system, particularly the innate immune system. We previously reported that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule CD1d to evade the antiviral function of NKT cells. Here we identified the key cellular motor protein KIF3A as a cellular substrate phosphorylated by US3, and this phosphorylation event mediates US3-induced immune evasion.

Burkholderia jirisanensis sp. nov., isolated from forest soil.

A Gram-negative, catalase-positive, mesophilic, obligately aerobic bacterium designated JRM2-1T was isolated from forest soil of Jirisan Mountain, Republic of Korea, and its taxonomic position was investigated based on a polyphasic taxonomic approach. Cells of strain JRM2-1T grew optimally at pH 5.0-7.0 and at 25 °C. Strain JRM2-1T was susceptible to chloramphenicol, gentamicin, kanamycin, nalidixic acid, rifampicin, streptomycin and tetracycline. On the basis of 16S rRNA gene sequence similarity, the closest neighbour of strain JRM2-1T was Burkholderia rhizosphaerae WR43T (98.1 %). On the basis of our phylogenetic analysis, strain JRM2-1T is clearly distinguished from related species of the genus Burkholderia and is clustered with plant-associated members of the genus. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The polar lipid profile of strain JRM2-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, several unidentified aminolipids and an unidentified aminophospholipid. The isoprenoid quinone of strain JRM2-1T was Q-8 and the DNA G+C content was 63.7 mol%. On the basis of our polyphasic taxonomic investigation, strain JRM2-1T is considered to represent a novel species in the genus Burkholderia, for which the name Burkholderia jirisanensis sp. nov. is proposed. The type strain is JRM2-1T ( = AIM 0373T = KCTC 42072T = JCM 19985T).

Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells.

Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vß8, the homolog of human Vß11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies.

Caproiciproducens galactitolivorans gen. nov., sp. nov., a bacterium capable of producing caproic acid from galactitol, isolated from a wastewater treatment plant.

A strictly anaerobic, Gram-stain-positive, non-spore-forming, rod-shaped bacterial strain, designated BS-1T, was isolated from an anaerobic digestion reactor during a study of bacteria utilizing galactitol as the carbon source. Its cells were 0.3-0.5 µm × 2-4 µm, and they grew at 35-45 °C and at pH 6.0-8.0. Strain BS-1T produced H2, CO2, ethanol, acetic acid, butyric acid and caproic acid as metabolic end products of anaerobic fermentation. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain BS-1T represented a novel bacterial genus within the family Ruminococcaceae, Clostridium Cluster IV. The type strains that were most closely related to strain BS-1T were Clostridium sporosphaeroides KCTC 5598T (94.5 %), Clostridium leptum KCTC 5155T (94.3 %), Ruminococcus bromii ATCC 27255T (92.1 %) and Ethanoligenens harbinense YUAN-3T (91.9 %). Strain BS-1T had 17.6 % and 20.9 % DNA-DNA relatedness values with C. sporosphaeroides DSM 1294T and C. leptum DSM 753T, respectively. The major components of the cellular fatty acids were C16 : 0 dimethyl aldehyde (DMA) (22.1 %), C16 : 0 aldehyde (14.1 %) and summed feature 11 (iso-C17 : 0 3-OH and/or C18 : 2 DMA; 10.0 %). The genomic DNA G+C content was 50.0 mol%. Phenotypic and phylogenetic characteristics allowed strain BS-1T to be clearly distinguished from other taxa of the genus Clostridium Cluster IV. On the basis of these data, the isolate is considered to represent a novel genus and novel species within Clostridium Cluster IV, for which the name Caproiciproducens galactitolivorans gen. nov., sp. nov. is proposed. The type species is BS-1T ( = JCM 30532T and KCCM 43048T).

Thermochemical hydrogen sensor based on chalcogenide nanowire arrays.

The hydrogen gas-sensing properties have been investigated of two types of thermochemical hydrogen (TCH) sensors composed of thermoelectric layers based on chalcogenide nanowire arrays and anodic aluminum oxide (AAO) templates. The monomorphic-type TCH sensor, which had only Bi2Te3 nanowire arrays, showed an output signal of 23.7 µV in response to 5 vol% hydrogen gas at room temperature, whereas an output signal of 215 µV was obtained from an n-p junction-type TCH sensor made of connected Bi2Te3 and Sb2Te3 nanowire arrays in an AAO template. Despite its small deposition area, the output signal of the n-p sensor was more than nine times that of the monomorphic sensor. This observation can be explained by the difference in electrical connections (parallel and serial conversions) in the TCH sensor between each type of nanowire array. Also, our n-p sensor had a wide detection range for hydrogen gas (from 400 ppm to 45 vol%) and a fast response time of 1.3 s at room temperature without requiring external power.

Facemask acne attenuation through modulation of indirect microbiome interactions.

During the COVID-19 pandemic, facemasks played a pivotal role in preventing person-person droplet transmission of viral particles. However, prolonged facemask wearing causes skin irritations colloquially referred to as 'maskne' (mask + acne), which manifests as acne and contact dermatitis and is mostly caused by pathogenic skin microbes. Previous studies revealed that the putative causal microbes were anaerobic bacteria, but the pathogenesis of facemask-associated skin conditions remains poorly defined. We therefore characterized the role of the facemask-associated skin microbiota in the development of maskne using culture-dependent and -independent methodologies. Metagenomic analysis revealed that the majority of the facemask microbiota were anaerobic bacteria that originated from the skin rather than saliva. Previous work demonstrated direct interaction between pathogenic bacteria and antagonistic strains in the microbiome. We expanded this analysis to include indirect interaction between pathogenic bacteria and other indigenous bacteria classified as either 'pathogen helper (PH)' or 'pathogen inhibitor (PIn)' strains. In vitro screening of bacteria isolated from facemasks identified both strains that antagonized and promoted pathogen growth. These data were validated using a mouse skin infection model, where we observed attenuation of symptoms following pathogen infection. Moreover, the inhibitor of pathogen helper (IPH) strain, which did not directly attenuate pathogen growth in vitro and in vivo, functioned to suppress symptom development and pathogen growth indirectly through PH inhibitory antibacterial products such as phenyl lactic acid. Taken together, our study is the first to define a mechanism by which indirect microbiota interactions under facemasks can control symptoms of maskne by suppressing a skin pathogen.

Asticcacaulis solisilvae sp. nov., isolated from forest soil.

An obligately aerobic, chemoheterotrophic, mesophilic prosthecate bacterium, designated strain CGM1-3EN(T), was isolated from the enrichment cultures of forest soil from Cheonggyesan Mountain, Republic of Korea. Cells were Gram-reaction-negative, motile rods (1.3-2.4 µm long by 0.30-0.75 µm wide) with single flagella. The strain grew at 10-37 °C (optimum 25-30 °C) and at pH 4.5-9.5 (optimum 5.0-7.0). The major cellular fatty acids were C16 : 0, C18 : 1ω7c 11-methyl, C12 : 1 3-OH and summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c). The genomic DNA G+C content of strain CGM1-3EN(T) was 63.7 mol%. The closest phylogenetic neighbour to strain CGM1-3EN(T) was identified as Asticcacaulis biprosthecium DSM 4723(T) (97.2 % 16S rRNA gene sequence similarity) and the DNA-DNA hybridization value between strain CGM1-3EN(T) and A. biprosthecium DSM 4723(T) was less than 24.5 %. Strain CGM1-3EN(T) used d-glucose, d-fructose, sucrose, maltose, trehalose, d-mannose, d-mannitol, d-sorbitol, d-galactose, cellobiose, lactose, raffinose, fumarate, pyruvate, dl-alanine and glycerol as carbon sources. Based on data from the present polyphasic study, the forest soil isolate CGM1-3EN(T) is considered to represent a novel species of the genus Asticcacaulis, for which the name Asticcacaulis solisilvae sp. nov. is proposed. The type strain is CGM1-3EN(T) ( = AIM0088(T) = KCTC 32102(T) = JCM 18544(T)).

Oscillibacter ruminantium sp. nov., isolated from the rumen of Korean native cattle.

A strictly anaerobic, Gram-negative, non-spore-forming bacterium, designated GH1(T), was isolated from the rumen of Korean native cattle (HanWoo). Cells were straight to slightly curved rods (2.0-4.5 µm long) and were motile by peritrichous flagella. The isolate grew at 30-45 °C (optimum 40 °C), at pH 5.5-6.5 (optimum pH 6.0) and with up to 3.5% (w/v) NaCl. Strain GH1(T) produced acid from d-glucose, d-ribose and d-xylose, with butyric acid being the major end product. The genomic DNA G+C content was 54.6 mol%. Based on comparative 16S rRNA gene sequence analysis, strain GH1(T) was most closely related to Oscillibacter valericigenes Sjm18-20(T) (97.3% 16S rRNA gene sequence similarity). DNA-DNA hybridization between strain GH1(T) and O. valericigenes DSM 18026(T) showed 24% reassociation. The major fatty acids were iso-C13:0 (13.0%), iso-C15:0 (17.6%), anteiso-C15:0 (8.4%) and C14:0 (4.1%), and the cellular fatty acid methyl esters as dimethylacetals (DMAs) were C16:0 DMA (17.8%), iso-C15:0 DMA (15.2%) and C14:0 DMA (4.52%). The cell-wall peptidoglycan of strain GH1(T) contained meso-diaminopimelic acid and the major cell-wall sugar was galactose. Based on 16S rRNA gene sequence similarity, phylogenetic analysis, DNA G+C content, DNA-DNA relatedness and distinct phenotypic characteristics, strain GH1(T) is classified in the genus Oscillibacter as a member of a novel species, for which the name Oscillibacter ruminantium sp. nov. is proposed. The type strain is GH1(T) (=KCTC 15176(T)=NBRC 108824(T)=JCM 18333(T)).

Conversion of levulinic acid to 2-butanone by acetoacetate decarboxylase from Clostridium acetobutylicum.

In this study, a novel system for synthesis of 2-butanone from levulinic acid (γ-keto-acid) via an enzymatic reaction was developed. Acetoacetate decarboxylase (AADC; E.C. 4.1.1.4) from Clostridium acetobutylicum was selected as a biocatalyst for decarboxylation of levulinic acid. The purified recombinant AADC from Escherichia coli successfully converted levulinic acid to 2-butanone with a conversion yield of 8.4-90.3 % depending on the amount of AADC under optimum conditions (30 °C and pH 5.0) despite that acetoacetate, a ß-keto-acid, is a natural substrate of AADC. In order to improve the catalytic efficiency, an AADC-mediator system was tested using methyl viologen, methylene blue, azure B, zinc ion, and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediators. Among them, methyl viologen showed the best performance, increasing the conversion yield up to 6.7-fold in comparison to that without methyl viologen. The results in this study are significant in the development of a renewable method for the synthesis of 2-butanone from biomass-derived chemical, levulinic acid, through enzymatic decarboxylation.

Microbial population dynamics and proteomics in membrane bioreactors with enzymatic quorum quenching.

Quorum sensing gives rise to biofilm formation on the membrane surface, which in turn causes a loss of water permeability in membrane bioreactors (MBRs) for wastewater treatment. Enzymatic quorum quenching was reported to successfully inhibit the formation of biofilm in MBRs through the decomposition of signal molecules, N-acyl homoserine lactones (AHLs). The aim of this study was to elucidate the mechanisms of quorum quenching in more detail in terms of microbial population dynamics and proteomics. Microbial communities in MBRs with and without a quorum quenching enzyme (acylase) were analyzed using pyrosequencing and compared with each other. In the quorum quenching MBR, the rate of transmembrane pressure (TMP) rise-up was delayed substantially, and the proportion of quorum sensing bacteria with AHL-like autoinducers (such as Enterobacter, Pseudomonas, and Acinetobacter) also decreased in the entire microbial community of mature biofilm in comparison to that in the control MBR. These factors were attributed to the lower production of extracellular polymeric substances (EPS), which are known to play a key role in the formation of biofilm. Proteomic analysis using the Enterobacter cancerogenus strain ATCC 35316 demonstrates the possible depression of protein expression related to microbial attachments to solid surfaces (outer membrane protein, flagellin) and the agglomeration of microorganisms (ATP synthase beta subunit) with the enzymatic quorum quenching. It has been argued that changes in the microbial population, EPS and proteins via enzymatic quorum quenching could inhibit the formation of biofilm, resulting in less biofouling in the quorum quenching MBR.

Enhancement of conductive pathway of functionalized CNT dispersed poly(methylmethacrylate) nanocomposites.

Poly(methylmethacrylate) (PMMA) beads were coated with pristine multi-walled carbon nanotube (MWNT) using various mixing medium and then nanocomposite were fabricated. Results were compared with acid functionalized MWNT (f-MWNT) coated PMMA bead nanocomposites. In addition, a combination of different mixing medium was also used to compare and to optimize the homogeneously dispersed MWNT and f-MWNT coating on PMMA beads. A homogeneous coating of nanotubes on the PMMA beads were observed in a DMF solution and confirmed by optical microscopy, SEM, Raman mapping, and sheet resistance measurements. Moreover, percolation and electrical properties were also compared with respect to nanotubes dispersion in the PMMA resin matrix. Nanocomposites prepared by coating PMMA beads with pristine CNT exhibited improvement of electro conductive pathway i.e., lowering percolation threshold below 0.1 wt% of MWNT content. The result was also compared with acid treated MWNT coated PMMA beads and acid treated dispersed MWNT in PMMA resins. The ultra-low electrical surface resistance of nanocomposites using trace amounts of MWNT coating on PMMA beads has not been reported so far.

Comparison of the bacterial communities in anaerobic, anoxic, and oxic chambers of a pilot A(2)O process using pyrosequencing analysis.

A(2)O process is a sequential wastewater treatment process that uses anaerobic, anoxic, and oxic chambers for nitrogen and phosphorus removal. In this study, the bacterial communities among these chambers were compared, and the diversity of the bacteria involved in nitrogen and phosphorus removal was surveyed. A pilot-scale A(2)O process (50 m(3) day(-1)) was operated for more than 6 months, and bacterial 16S rRNA gene diversity was analyzed using pyrosequencing. A total of 7,447 bacterial sequence reads were obtained from anaerobic (1,546), anoxic (2,158), and oxic (3,743) chambers. Even though there were differences in the atmospheric condition and functionality, no prominent differences could be found in the bacterial community of the three chambers of the pilot A(2)O process. All sequence reads, which were taxonomically analyzed using the Eztaxon-e database, were assigned into 638 approved or tentative genera. Among them, about 72.2 % of the taxa were contained in the phyla Proteobacteria and Bacteroidetes. Phosphate-accumulating bacteria, Candidatus Accumulibacter phosphatis, and two other Accumulibacter were found to constitute 3.1 % of the identified genera. Ammonia-oxidizing bacteria, Nitrosomonas oligotropha, and four other phylotypes in the same family, Nitrosomonadaceae, constituted 0.2 and 0.9 %, respectively. Nitrite-oxidizing bacteria, Nitrospira defluvii, and other three phylotypes in the same family, Nitrospiraceae, constituted 2.5 and 0.1 %, respectively. In addition, Dokdonella and a phylotype of the phylum Chloroflexi, function in nitrogen and/or phosphate removal of which have not been reported in the A(2)O process, constituted the first and third composition among genera at 4.3 and 3.8 %, respectively.