A novel bioprospecting strategy via 13C-based high-throughput probing of active methylotrophs inhabiting oil reservoir surface soil.

Methane-oxidizing bacteria (MOB) have long been considered as a microbial indicator for oil and gas prospecting. However, due to the phylogenetically narrow breath of ecophysiologically distinct MOB, classic culture-dependent approaches could not discriminate MOB population at fine resolution, and accurately reflect the abundance of active MOB in the soil above oil and gas reservoirs. Here, we presented a novel microbial anomaly detection (MAD) strategy to quantitatively identify specific indicator methylotrophs in the surface soils for bioprospecting oil and gas reservoirs by using a combination of 13C-DNA stable isotope probing (SIP), high-throughput sequencing (HTS), quantitative PCR (qPCR) and geostatistical analysis. The Chunguang oilfield of the Junggar Basin was selected as a model system in western China, and type I methanotrophic Methylobacter was most active in the topsoil above the productive oil wells, while type II methanotrophic Methylosinus predominated in the dry well soils, exhibiting clear differences between non- and oil reservoir soils. Similar results were observed by quantification of Methylobacter pmoA genes as a specific bioindicator for the prediction of unknown reservoirs by grid sampling. A microbial anomaly distribution map based on geostatistical analysis further showed that the anomalous zones were highly consistent with petroleum, geological and seismic data, and validated by subsequent drilling. Over seven years, a total of 24 wells have been designed and drilled into the targeted anomaly, and the success rate via the MAD prospecting strategy was 83 %. Our results suggested that molecular techniques are powerful tools for oil and gas prospecting. This study indicates that the exploration efficiency could be significantly improved by integrating multi-disciplinary information in geophysics and geomicrobiology while reducing the drilling risk to a greater extent.

Identifying Active Rather than Total Methanotrophs Inhabiting Surface Soil Is Essential for the Microbial Prospection of Gas Reservoirs.

Methane-oxidizing bacteria (MOB) have long been recognized as an important bioindicator for oil and gas exploration. However, due to their physiological and ecological diversity, the distribution of MOB in different habitats varies widely, making it challenging to authentically reflect the abundance of active MOB in the soil above oil and gas reservoirs using conventional methods. Here, we selected the Puguang gas field of the Sichuan Basin in Southwest China as a model system to study the ecological characteristics of methanotrophs using culture-independent molecular techniques. Initially, by comparing the abundance of the pmoA genes determined by quantitative PCR (qPCR), no significant difference was found between gas well and non-gas well soils, indicating that the abundance of total MOB may not necessarily reflect the distribution of the underlying gas reservoirs. 13C-DNA stable isotope probing (DNA-SIP) in combination with high-throughput sequencing (HTS) furthermore revealed that type II methanotrophic Methylocystis was the absolutely predominant active MOB in the non-gas-field soils, whereas the niche vacated by Methylocystis was gradually filled with type I RPC-2 (rice paddy cluster-2) and Methylosarcina in the surface soils of gas reservoirs after geoscale acclimation to trace- and continuous-methane supply. The sum of the relative abundance of RPC-2 and Methylosarcina was then used as specific biotic index (BI) in the Puguang gas field. A microbial anomaly distribution map based on the BI values showed that the anomalous zones were highly consistent with geological and geophysical data, and known drilling results. Therefore, the active but not total methanotrophs successfully reflected the microseepage intensity of the underlying active hydrocarbon system, and can be used as an essential quantitative index to determine the existence and distribution of reservoirs. Our results suggest that molecular microbial techniques are powerful tools for oil and gas prospecting.

Synergism Between Multi-Pseudomonas and Cutinase for Biodegradation of Crude Oil-Based Derivatives.

Polyethylene terephthalate (PET) as one of the main crude oil-based derivatives, produces a significant amount of waste that is difficult to degrade. Currently, microbial degradation of PET is an eco-friendly, efficient, and economical method. This study was conducted to propose a novel screening strategy for PET-degrading bacteria, and evaluate their degradation efficiency of PET. Two strains, Pseudomonas nitroreducens S8 and Pseudomonas monteilii S17, were isolated and could utilize PET as a carbon source by co-culture. The combined use of both bacteria gave a synergistic effect on the disruption of the PET surface through colonization behavior, which could enhance the subsequent degradation of PET. Its time of reaching a peak value of PET degradation rate (94.5% at 6 d) was 2 days earlier than these of single bacteria. A similar synergistic effect was also observed in the metabolization of PET monomers, and the metabolic rate was expressed as 82.4% of bis (2-hydroxyethyl) terephthalate (BHET), 64.0% of mono (2-hydroxyethyl) terephthalate (MHET), and 20.0% of terephthalic acid (TPA), respectively. This study is novel in showing the degradation of PET waste by combinations of bacterial pretreatment and enzymatic treatment, which can be a promising method.

Synergistic biodegradation of poly(ethylene terephthalate) using Microbacterium oleivorans and Thermobifida fusca cutinase.

Poly(ethylene terephthalate) (PET) is a major source of plastic pollution. Biodegradation technologies are of paramount interest in reducing or recycling PET waste. In particular, a synergistic microbe-enzyme treatment may prove to be a promising approach. In this study, a synergistic system composed of Microbacterium oleivorans JWG-G2 and Thermobifida fusca cutinase (referred to as TfC) was employed to degrade bis(hydroxyethyl) terephthalate (BHET) oligomers and a high crystalline PET film. A novel degradation product that was obtained by M. oleivorans JWG-G2 treatment alone was identified as ethylene glycol terephthalate (EGT). With the addition of TfC as a second biocatalyst, the highest synergy degrees for BHET oligomers and PET film degradation were 2.79 and 2.26, respectively. The largest amounts of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) (47 nM and 330 nM, respectively) were detected after combined treatment of PET film with M. oleivorans JWG-G2 at 5 × 103 µL/cm2 and TfC at 120 µg/cm2, and the degree of PET film surface destruction was more significant than those produced by each treatment alone. The presence of extracellular PET hydrolases in M. oleivorans JWG-G2, including three carboxylesterases, an esterase and a lipase, was predicted by whole genome sequencing analysis, and a predicted PET degradation pathway was proposed for the synergistic microbe-enzyme treatment. The results indicated that synergistic microbe-enzyme treatment may serve as a potentially promising tool for the future development of effective PET degradation. KEY POINTS: • An ecofriendly synergistic microbe-enzyme PET degradation system operating at room temperature was first introduced for degrading PET. • A novel product (EGT) was first identified during PET degradation. • Potential PET hydrolases in M. oleivorans JWG-G2 were predicted by whole genome sequencing analysis.

A dual-functional aminopeptidase from Streptomyces canus T20 and its application in the preparation of small rice peptides.

An aminopeptidase that derived from Streptomyces canus T20 (ScAP) was successfully expressed and characterized in Escherichia coli BL21 (DE3) for first time. The specific activity was 6000 U/mg, which is highest in Streptomyces aminopeptidases. Its optimal conditions were 60 °C and pH 8.0, respectively. ScAP exhibited excellent thermal and alkaline pH stability, retained 80.0% maximal activity at 50 °C for 200 h or at pH 9.0 for 24 h. Its activity observed to be complete inhibited by 0.1 mM EDTA and enhanced by Ca2+ and Co2+ to 115.4% and 104.0% respectively. ScAP also has exhibited high specificity towards rice protein on preparation of small peptides. The yield of small rice peptides achieved 66.5%, which is highest by far. Besides, ScAP have significant debittering effect on rice peptides. Results showed that bitter intensity score decreased by 49.0% with optimum condition (0.048% ScAP at 50 °C for 6 h). Therefore, ScAP as dual functional aminopeptidase of hydrolytic and debittering might have a potential application in the production of high yield and low bitterness of small rice peptides.

Rubus idaeus L. (red raspberry) blocks UVB-induced MMP production and promotes type I procollagen synthesis via inhibition of MAPK/AP-1, NF-κß and stimulation of TGF-ß/Smad, Nrf2 in normal human dermal fibroblasts.

Chronic exposure to ultraviolet (UV) radiation causes photo-oxidation, which in turn results in the upregulation of matrix metalloproteinases (MMPs) and loss of collagen. Rubus idaeus L. (RI), also called red raspberry, is an important cash crop that contains abundant antioxidant compounds. Sanguiin H-6 and lambertianin C are the major ingredients presented in the extracts. Here, we studied the protective effect of RI on UVB-induced photoaging in normal human dermal fibroblasts (NHDFs). We found that RI notably reduced UVB-induced MMPs secretion and pro-inflammatory mediators production, and significantly suppressed UVB-induced activation of mitogen-activated protein kinase (MAPK), nuclear factor-κß, as well as activator protein 1. Additionally, treatment of NHDFs with the ERK inhibitor (PD98059) and JNK inhibitor (SP600125) resulted in the reduction of UVB-induced MMP-1 and IL-6 expressions, which demonstrated that the inhibition of MMP-1 and IL-6 by RI is associated with the MAPK pathway. Furthermore, we also found that RI accelerated procollagen type I synthesis by activating the transforming growth factor-ß/Smad pathway and enhanced the expression of cytoprotective antioxidants such as heme oxygenase-1 and NHD(P)H quinone oxidoreductase 1 by promoting nuclear factor E2-related factor 2 nuclear transfer. Overall, these findings demonstrated that RI was potentially effective in preventing UVB induced skin photoaging.

Nibribacter flagellatus sp. nov., isolated from rhizosphere of Hibiscus syriacus and emended description of the genus Nibribacter.

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10-40 °C (optimum 28-30 °C), at pH 6.0-8.0 (optimum 7.0) and at 0-1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16T were identified as Nibribacter koreensis KACC 16450T (98.6%), Rufibacter roseus KCTC 42217T (94.7%), Rufibacter immobilis CCTCC AB 2013351T (94.5%) and Rufibacter tibetensis CCTCC AB 208084T (94.4%). The DNA G+C content of strain THG-T16T was determined to be 46.7 mol%. DNA-DNA hybridization values between strain THG-T16T and N. koreensis KACC 16450T, R. roseus KCTC 42217T, R. immobilis CCTCC AB 2013351T, R.tibetensis CCTCC AB 208084T were 33.5 ± 0.5% (31.7 ± 0.7% reciprocal analysis), 28.1 ± 0.2% (25.2 ± 0.2%), 17.1 ± 0.9% (10.2 ± 0.6%) and 8.1 ± 0.3% (5.2 ± 0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C16:1 ω5c, C17:1 ω6c, iso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA-DNA hybridization data, strain THG-T16T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16T(= KACC 19188T = CCTCC AB 2016246T).

Paracoccus pueri sp. nov., isolated from Pu'er tea.

A Gram-stain negative, aerobic, short rod-shaped, motile by flagella bacterial strain (THG-N2.35T), was isolated from Pu'er tea. Growth occurred at 10-40 °C (optimum 28 °C), at pH 4-7 (optimum 7) and at 0-5% NaCl (optimum 1%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-N2.35T were identified as Paracoccus hibisci KACC 18632T (99.0%), Paracoccus tibetensis CGMCC 1.8925T (98.7%), Paracoccus beibuensis CGMCC 1.7295T (98.2%), Paracoccus aestuarii KCTC 22049T (98.2%), Paracoccus rhizosphaerae LMG 26205T (98.1%), Paracoccus zeaxanthinifaciens ATCC 21588T (97.1%), Paracoccus marcusii DSM 11574T (97.0%). Levels of similarity between strain THG-N2.35T and other Paracoccus species were lower than 97.0%. DNA-DNA hybridization values between strain THG-N2.35T and P. hibisci KACC 18632T, P. tibetensis CGMCC 1.8925T, P. beibuensis CGMCC 1.7295T, P. aestuarii KCTC 22049T, P. rhizosphaerae LMG 26205T, P. zeaxanthinifaciens ATCC 21588T, P.marcusii DSM 11574T were 47.5% (42.3%, reciprocal analysis), 36.1% (32.3%), 24.7% (22.1%), 19.2% (16.3%), 11.3% (8.8%), 11.1% (10.8%), 6.1% (5.8%), respectively. The DNA G+C content of strain THG-N2.35T was 62.3 mol%. The polar lipids were diphosphatidylglycerol, phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The quinone was ubiquinone-10 (Q-10). The major fatty acids were C10:0 3OH, C16:0, C18:0 and C18:1 ω7ϲ. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-N2.35T represent a novel species of the genus Paracoccus, for which the name Paracoccus pueri sp. nov. is proposed. The type strain is THG-N2.35T (= KACC 18934T = CCTCC AB 2016177T).

Pterocarpus santalinus L. Regulated Ultraviolet B Irradiation-induced Procollagen Reduction and Matrix Metalloproteinases Expression Through Activation of TGF-ß/Smad and Inhibition of the MAPK/AP-1 Pathway in Normal Human Dermal Fibroblasts.

Ultraviolet light-induced reactive oxygen species (ROS) damage human skin and prematurely cause aging. A growing body of research is focusing on considering plants and plant-derived compounds as antiphotoaging therapeutic material. Pterocarpus santalinus L., as an Indian traditional medicine, possesses antidiabetic, anti-inflammatory and antioxidative effects. Here, we studied the antiphotoaging effects of ethanolic extract of P. santalinus L. heartwood (EPS) on ultraviolet radiation B (UVB)-irradiated normal human dermal fibroblasts (NHDFs). Results showed that EPS significantly inhibited the upregulation of matrix metalloproteinases and IL-6 caused by UVB irradiation, and suppressed UVB-induced phosphorylation of extracellular signal-regulated kinase, Jun N-terminal kinase and p38, as well as the activation of AP-1 transcription factors. Further study indicated that UVB-induced production of MMP-1 and IL-6 could be inhibited by PD 98059 (an ERK inhibitor) and SP600125 (A JNK inhibitor), implied that EPS inhibited UVB-induced MMP-1 and IL-6 secretion by inactivating MAPK signaling pathway. In addition, EPS possessed an excellent antioxidant activity, which could increase cytoprotective antioxidants such as HO-1, NQ-O1 expression by facilitating the nuclear accumulation of Nrf2. Treatment of NHDFs with EPS also recovered UVB-induced procollagen type I reduction by activating TGF-ß/Smad pathway. These findings demonstrated that EPS had a potential effect against UVB-induced skin photoaging.

Deinococcus hibisci sp. nov., isolated from rhizosphere of Hibiscus syriacus L. (mugunghwa flower).

A Gram-stain-positive, pink-pigmented, coccus-shaped, strictly aerobic, non-motile bacterium, strain THG-AG1.5T, was isolated from rhizosphere of Hibiscus syriacus L. (Mugunghwa flower) located in Kyung Hee University, Yongin, Gyeonggi, Republic of Korea. The isolated strain grew optimally at 25-30 °C, at pH 6.0-7.5 and in the presence of additional 0-1.5 % (w/v) NaCl. Strain THG-AG1.5T exhibited tolerance to UV radiation (>1500 J m-2) and to gamma radiation (>12 kGy). Based on 16S rRNA gene sequence comparisons, strain THG-AG1.5T was closely related to Deinococcus daejeonensis MJ27T (98.03 %), Deinococcus radiotolerans C1T (97.61 %) and Deinococcus grandis DSM 3963T (97.32 %). The genomic DNA G+C content of strain THG-AG1.5T was 74.8 mol%. The DNA-DNA hybridization values between strain THG-AG1.5T and its closest phylogenetically neighbours were below 63.0 %. The peptidoglycan amino acids were alanine, valine, glutamic acid, glycine, ornithine, lysine and aspartic acid. Strain THG-AG1.5T contained ribose, mannose and glucose as whole-cell-wall sugars and menaquinone-8 (MK-8) as the only isoprenoid quinone. The major component in the polyamine pattern was spermidine. The major polar lipids of strain THG-AG1.5T were a phosphoglycolipid, six unidentified glycolipids and an unidentified aminophospholipid. The major fatty acids were identified as iso-C15 : 0, C15 : 1ω6c, C16 : 0, iso-C17 : 0, C17 : 0, C18 : 0 and summed feature 3. On the basis of our polyphasic taxonomy study, strain THG-AG1.5T represents a novel species within the genus Deinococcus, for which the name Deinococcushibisci sp. nov. is proposed. The type strain is THG-AG1.5T (=KACC 18850T=CCTCC AB 2016078T).

Corynebacterium defluvii sp. nov., isolated from Sewage.

A Gram-positive, aerobic, non-motile, rod-shapeds, catalase-positive, and oxidase-negative strain, designated Y49T, was isolated from sewage collected from Jilin Agricultural University, China. It grew at 20-40°C (optimum at 30°C), at pH 6.0-8.0 (optimum at 7.0) and at 0-1.0% sodium chloride (optimum at 0%). The major isoprenoid quinone was menaquinone-8 (MK-8) and the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, four unidentified lipids, and two unidentified aminolipids. The peptidoglycan was meso-diaminopimelic acid. The cell-wall sugars were galactose, arabinose, and glucose. The fatty acids were C9:0, C16:0, C16:1 ω9c, C17:1 ω9c, C18:3 ω6c (6,9,12), C18:1 ω9c, and C18:0. The DNA G+C content was 51.4 mol%. Based on the 16S rRNA gene sequence analysis, the nearest phylogenetic neighbors of strain Y49T were Corynebacterium efficiens DSM 44549T (97.5%), Corynebacterium callunae DSM 20147T (97.2%), Corynebacterium deserti GIMN 1.010T (96.8%), Corynebacterium glutamicum ATCC 13032T (96.4%), and other species belonging to this genus (92.3-95.4%). The DNA-DNA relatedness value between strain Y49T and C. efficiens DSM 44549T, C. callunae DSM 20147T, C. deserti GIMN1.010T, and C. glutamicum ATCC 13032T was 25.5±2.0%, 21.1±1.0%, 16.5±0.5%, and 13.5±0.9%, respectively. Based on the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain Y49T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium defluvii sp nov. is proposed. The type strain is Y49T (= KCTC 39731T =CGMCC 1.15506T).

Aeromicrobium halotolerans sp. nov., isolated from desert soil sample.

A Gram-positive, aerobic, and non-motile, rod-shaped actinomycete strain, designated YIM Y47(T), was isolated from soils collected from Turpan desert, China, and subjected to a polyphasic taxonomic study. Phylogenetic analysis indicated that strain YIM Y47(T) belonged to the genus Aeromicrobium. YIM Y47(T) shared highest 16S rRNA gene sequence similarities with Aeromicrobium massiliense JC14(T) (96.47 %). Growth occurs at 20-45 °C (optimum at 30 °C), pH 6.0-8.0 (optimum at pH 7.0), and salinities of 0-7.0 % NaCl (optimum at 4.0 %). The strain YIM Y47(T) exhibits chemotaxonomic features with menaquinone-7 (MK-7) as the predominant quinone, C16:0, C18:1 ω9c and 10-methyl C18:0 (>10 %) as major fatty acids. The cell-wall peptidoglycan of strain YIM Y47(T) contained LL-diaminopimelic acid as the diagnostic diamino acid. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and unknown phospholipids. The G+C content of the genomic DNA of strain YIM Y47(T) was found to be 44.7 mol%. On the basis of phylogenetic analyses and phenotypic data, it is proposed that strain YIM Y47(T) should be classified as representing a novel species of the genus Aeromicrobium, with the name Aeromicrobium halotolerans sp. nov. The type strain is YIM Y47(T) (=KCTC 39113(T)=CGMCC 1.15063(T)=DSM 29939(T)=JCM 30627(T)).

Inhibitory and Acceleratory Effects of Inonotus obliquus on Tyrosinase Activity and Melanin Formation in B16 Melanoma Cells.

The aim of the present study is to preliminarily investigate the antimelanogenesis effect of Inonotus obliquus extracts by cell-free mushroom tyrosinase assay. It was found that petroleum ether and n-butanol extracts might contain unknown potential tyrosinase inhibitors, while its ethyl acetate extract might contain some unknown accelerators. Six compounds were isolated and their structures were identified by interpretation of NMR data and nicotinic acid was first discovered in Inonotus obliquus. In cells testing, betulin and trametenolic acid decreased tyrosinase activity and melanin content, while inotodiol and lanosterol significantly increased tyrosinase activity and melanin content, showing an AC⁡50 of 9.74 and 8.43 µM, respectively. Nicotinie acid, 3ß,22,25-trihydroxy-lanosta-8-ene, had a little or no effect on tyrosinase. Betulin exhibited a mode of noncompetitive inhibition with a K I = K IS of 0.4 µM on tyrosinase activity showing an IC50 of 5.13 µM and being more effective than kojic acid (6.43 µM), and trametenolic acid exhibited a mode of mixed inhibition with a K I of 0.9 µM, K IS of 0.5 µM, and an IC50 of 7.25 µM. We proposed betulin and trametenolic acid as a new candidate of potent tyrosinase inhibitors and inotodiol and lanosterol as accelerators that could be used as therapeutic agent.

Activation effects of polysaccharides of Flammulina velutipes mycorrhizae on the T lymphocyte immune function.

Flammulina velutipes mycorrhizae have increasingly been produced with increasing of F. velutipes production. A mouse model was thus used to examine potential effect of F. velutipes mycorrhizae on the immune function. Fifty female Wistar mice (5-weeks-old) weighed 15-20 g were randomly allocated into five groups. Polysaccharide of F. velutipes mycorrhizae were treated with mice and mice spleen lymphocytes. The levels of CD3(+), CD4(+), and CD8(+) T lymphocyte, interleukin-2 (IL-2), and tumor necrosis factor-a (TNF-α) were determined. The results showed that the proportions of CD3(+), and CD4(+) T lymphocyte, the ratio of CD4(+)/CD8(+), and the levels of IL-2 and TNF-a were significantly increased in polysaccharide of F. velutipes mycorrhizae, while the proportion of CD8(+) T lymphocyte was decreased in polysaccharide of F. velutipes mycorrhizae-dose dependent manner. Our findings indicated that a long term exposure of polysaccharide of F. velutipes mycorrhizae could activate the T lymphocyte immune function. Polysaccharide of F. velutipes mycorrhizae was expected to develop into the immune health products.