Illumina MiSeq Sequencing Reveals Diverse Microbial Communities of Activated Sludge Systems Stimulated by Different Aromatics for Indigo Biosynthesis from Indole.
ABSTRACT: Indole, as a typical N-heteroaromatic compound existed in coking wastewater, can be used for bio-indigo production. The microbial production of indigo from indole has been widely reported during the last decades using culture-dependent methods, but few studies have been carried out by microbial communities. Herein, three activated sludge systems stimulated by different aromatics, i.e. naphthalene plus indole (G1), phenol plus indole (G2) and indole only (G3), were constructed for indigo production from indole. During the operation, G1 produced the highest indigo yield in the early stage, but it switched to G3 in the late stage. Based on LC-MS analysis, indigo was the major product in G1 and G3, while the purple product 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one was dominant in G2. Illumina MiSeq sequencing of 16S rRNA gene amplicons was applied to analyze the microbial community structure and composition. Detrended correspondence analysis (DCA) and dissimilarity tests showed that the overall community structures of three groups changed significantly during the operation (P<0.05). Nevertheless, the bacteria assigned to phylum Proteobacteria, family Comamonadaceae, and genera Diaphorobacter, Comamonas and Aquamicrobium were commonly shared dominant populations. Pearson correlations were calculated to discern the relationship between microbial communities and indigo yields. The typical indigo-producing populations Comamonas and Pseudomonas showed no positive correlations with indigo yields, while there emerged many other genera that exhibited positive relationships, such as Aquamicrobium, Truepera and Pusillimonas, which had not been reported for indigo production previously. The present study should provide new insights into indigo bio-production by microbial communities from indole.
Project description:Biosynthesis of the popular dyestuff indigo from indole has been comprehensively studied using pure cultures, but less has been done to characterize the indigo production by microbial communities. In our previous studies, a wild strain Comamonas sp. MQ was isolated from activated sludge and the recombinant Escherichia coli nagAc carrying the naphthalene dioxygenase gene (nag) from strain MQ was constructed, both of which were capable of producing indigo from indole. Herein, three activated sludge systems, G1 (non-augmented control), G2 (augmented with Comamonas sp. MQ), and G3 (augmented with recombinant E. coli nagAc), were constructed to investigate indigo production. After 132-day operation, G3 produced the highest yields of indigo (99.5 ± 3.0 mg/l), followed by G2 (27.3 ± 1.3 mg/l) and G1 (19.2 ± 1.2 mg/l). The microbial community dynamics and activities associated with indigo production were analyzed by Illumina Miseq sequencing of 16S rRNA gene amplicons. The inoculated strain MQ survived for at least 30 days, whereas E. coli nagAc was undetectable shortly after inoculation. Quantitative real-time PCR analysis suggested the abundance of naphthalene dioxygenase gene (nagAc) from both inoculated strains was strongly correlated with indigo yields in early stages (0-30 days) (P < 0.001) but not in later stages (30-132 days) (P > 0.10) of operation. Based on detrended correspondence analysis (DCA) and dissimilarity test results, the communities underwent a noticeable shift during the operation. Among the four major genera (> 1% on average), the commonly reported indigo-producing populations Comamonas and Pseudomonas showed no positive relationship with indigo yields (P > 0.05) based on Pearson correlation test, while Alcaligenes and Aquamicrobium, rarely reported for indigo production, were positively correlated with indigo yields (P < 0.05). This study should provide new insights into our understanding of indigo bio-production by microbial communities.
Project description:Breakdown of plant biomass in rumen depends on interactions between bacteria, archaea, fungi, and protozoa; however, the majority of studies of the microbiome of ruminants, including the few studies of the rumen of camels, only studied one of these microbial groups. In this study, we applied total rRNA sequencing to identify active microbial communities in 22 solid and liquid rumen samples from 11 camels. These camels were reared at three stations that use different feeding systems: clover, hay and wheat straw (G1), fresh clover (G2), and wheat straw (G3). Bacteria dominated the libraries of sequence reads generated from all rumen samples, followed by protozoa, archaea, and fungi respectively. Firmicutes, Thermoplasmatales, Diplodinium, and Neocallimastix dominated bacterial, archaeal, protozoal and fungal communities, respectively in all samples. Libraries generated from camels reared at facility G2, where they were fed fresh clover, showed the highest alpha diversity. Principal co-ordinate analysis and linear discriminate analysis showed clusters associated with facility/feed and the relative abundance of microbes varied between liquid and solid fractions. This provides preliminary evidence that bacteria dominate the microbial communities of the camel rumen and these communities differ significantly between populations of domesticated camels.
Project description:The grass parasitic fungus Claviceps purpurea sensu lato produces sclerotia with toxic indole alkaloids. It constitutes several genetic groups with divergent habitat preferences that recently were delimited into separate proposed species. We aimed to 1) analyze genetic variation of C. purpurea sensu lato in Norway, 2) characterize the associated indole alkaloid profiles, and 3) explore relationships between genetics, alkaloid chemistry and ecology. Approximately 600 sclerotia from 14 different grass species were subjected to various analyses including DNA sequencing and HPLC-MS. Molecular results, supported by chemical and ecological data, revealed one new genetic group (G4) in addition to two of the three known; G1 (C. purpurea sensu stricto) and G2 (C. humidiphila). G3 (C. spartinae) was not found. G4, which was apparently con-specific with the recently described C. arundinis sp. nov, was predominantly found in very wet habitats on Molinia caerulea and infrequently in saline habitats on Leymus arenarius. Its indole-diterpene profile resembled G2, while its ergot alkaloid profile differed from G2 in high amounts of ergosedmam. In contrast to G1, indole-diterpenes were consistently present in G2 and G4. Our study supports and complements the newly proposed species delimitation of the C. purpurea complex, but challenges some species characteristics including host spectrum, habitat preferences and sclerotial floating ability.
Project description:Indole is widely spread in various environmental matrices. Indole degradation by bacteria has been reported previously, whereas its degradation processes driven by aerobic microbial community were as-yet unexplored. Herein, eight sequencing batch bioreactors fed with municipal and coking activated sludges were constructed for aerobic treatment of indole. The whole operation processes contained three stages, i.e. stage I, glucose and indole as carbon sources; stage II, indole as carbon source; and stage III, indole as carbon and nitrogen source. Indole could be completely removed in both systems. Illumina sequencing revealed that alpha diversity was reduced after indole treatment and microbial communities were significantly distinct among the three stages. At genus level, Azorcus and Thauera were dominant species in stage I in both systems, while Alcaligenes, Comamonas and Pseudomonas were the core genera in stage II and III in municipal sludge system, Alcaligenes and Burkholderia in coking sludge system. In addition, four strains belonged to genera Comamonas, Burkholderia and Xenophilus were isolated using indole as sole carbon source. Burkholderia sp. IDO3 could remove 100?mg/L indole completely within 14?h, the highest degradation rate to date. These findings provide novel information and enrich our understanding of indole aerobic degradation processes.
Project description:The study was conducted to optimize lighting schedule for pre-pubertal (12 to 22 weeks) Chinese native breed Pengxian yellow pullet. A total of 414 healthy pullets (10 weeks), with similar body weight were randomly distributed into three groups (n = 138) and housed in individual cages for up to 12 weeks of age in light controlled rooms and provided normal lighting schedule (10L:14D). At 12 to 18 weeks of age, pullets were housed in three rooms, having varying lighting schedule viz. G1 (8L: 16D), G2 (10L:14D), or G3 (12L:12D). From 19th week onwards lighting schedule was gradually increased every week in incremental manner till all groups started receiving 16L:8D lighting schedule. The age at first egg, weight of first egg laid, percent peak hen day egg production, concentration of plasma luteinizing and follicle-stimulating hormones and expression of genes regulating synthesis or/and secretion of hypothalamic gonadotropin-releasing hormone-I (GnRH-I), and pituitary LH-? and FSH-? were studied during experimental period (12 to 43 weeks of age) of this study. The result indicated that pullets of long day length (G3) group had higher plasma levels of FSH and LH and also better mRNA expression that regulates synthesis or/and secretion of GnRH-I, FSH-?, and LH-? before egg laying. The age at first egg (151.3 days) in pullets of G3 group receiving longer lighting hours (12L:12D) was 8.8 days less (P<0.05) compared to pullets of G1 group, while it was 6.9 days less (P>0.05) compared to G2. However, significantly higher (P<0.05) plasma levels of LH and FSH in pullets of G1 as compared to pullets belonging to G3 group corresponded with the higher (P<0.05) cumulative egg production during the experimental period, while these attributes in G2 group didn't differ from either G1 or G3 groups. Pullets of G1 group had significantly higher levels (P<0.05) of GnRH-I, FSH-?, and LH-? mRNA abundances at 43 weeks of age than other two groups and this corresponded with the percent (hen day) peak egg production (75.38%) in pullets in this G1 group that was attained at 32 weeks of age, while the peak production of 71.24% was attained at 30 weeks of age in G3 group. There was no effect of lighting schedule on body weight of pullets, recorded during experimental period, at all occasions; belonging to three groups (G1,G2 and G3) and receiving varying hours of photo-stimulation (P>0.05). It was inferred that the optimum lighting schedule for Chinese native breed Pengxian yellow pullets during 10 weeks of pre-pubertal growth period is short hours of photo-stimulation (i.e 8L:16D).
Project description:Using prospective longitudinal data from three generations, this study seeks to test whether and how parent and grandparent marijuana use (current and prior) predicts an increased likelihood of child cigarette, alcohol, and marijuana use.Using multilevel modeling of prospective data spanning three generations (n = 306 families, children ages 6-22), this study tested associations between grandparent (G1) and parent (G2) marijuana use and child (G3) past-year cigarette, alcohol, and marijuana use. Analyses tested whether G3 substance-related norms mediated these associations. Current G1 and G2 marijuana use was examined, as was G2 high school and early adult use and G1 marijuana use when G2 parents were in early adolescence. Controls included G2 age at G3 birth, G2 education and depression, and G3 gender.G2 current marijuana use predicted a higher likelihood of G3 alcohol and marijuana use but was not related to the probability of G3 cigarette use. G3's perceptions of their parents' norms and G2 current marijuana use both contributed independently to the likelihood of G3 alcohol and marijuana use when included in the same model. G3 children's own norms and their perceptions of friends' norms mediated the link between G2 current marijuana use and G3 alcohol and marijuana use.Results are discussed in light of the growing trend toward marijuana legalization. To the extent that parent marijuana use increases under legalization, we can expect more youth to use alcohol and marijuana and to have norms that favor substance use.
Project description:AIM:Charcot foot (CF) is a rare complication of Type 2 diabetes (T2D). MATERIALS & METHODS:We assessed circulating miRNAs in 17 patients with T2D and acute CF (G1), 17 patients with T2D (G2) and equivalent neuropathy and 17 patients with T2D without neuropathy (G3) using the high-throughput miRNA expression profiling. RESULTS:51 significantly deregulated miRNAs were identified in G1 versus G2, 37 in G1 versus G3 and 64 in G2 versus G3. Furthermore, we demonstrated that 16 miRNAs differentially expressed between G1 versus G2 could be involved in osteoclastic differentiation. Among them, eight are key factors involved in CF pathophysiology. CONCLUSION:Our data reveal that CF patients exhibit an altered expression profile of circulating miRNAs.
Project description:Method Data of patients who were surgically treated and clinicopathologically diagnosed as (MH)-NENs secondary to (GEP)-NENs at West China Hospital of Sichuan University from January 2006 to December 2018 were retrospectively collected and analyzed by the grading classification for (GEP)-NENs. Results We identified 150 patients with (MH)-NENs secondary to (GEP)-NENs, including 10 patients with G1 NETs, 26 with G2 NETs, 33 with G3 NETs, and 81 with G3 NECs. There were significant differences between patients with G1/G2/G3 NETs and those with G3 NECs, such as age at diagnosis (P=0.041), synchronous liver lesion (P=0.032), incidental diagnosis (P=0.014), tumor largest diameter (P=0.047), vascular invasion (P=0.017), and extrahepatic metastatic disease (P=0.029). The estimated 3-year overall survival for patients with G1 NETs, G2 NETs, G3 NETs, and G3 NECs was 100%, 79.4%, 49.5%, and 20.7%, respectively (P < 0.001). The survival of G1 NETs or G2 NETs was significantly better than that of G3 NETs (P=0.013, P=0.037, respectively) and G3 NECs (P=0.001, P < 0.001; respectively). Patients with G3 NECs present notably worse survival than those with G3 NETs (P=0.012), while survival comparison between G1 NETs and G2 NETs was not statistically different (P=0.131). The grading classification for (GEP)-NENs was an effective independent predictor of survival for (MH)-NENs secondary to (GEP)-NENs (hazard ratio: 4.234; 95% confidence intervals: 1.984–6.763; P=0.003). Conclusion Our demonstration revealed that the grading classification for (GEP)-NENs could well stratify (MH)-NENs secondary to (GEP)-NENs into prognostic groups and supported its wide use in clinical practice.
Project description:This study investigated the effect of CO2 laser irradiation on the inhibition of secondary caries on root surfaces adjacent to glass ionomer cement (GIC) or composite resin (CR) restorations. 40 dental blocks were divided into 4 groups: G1 (negative control): cavity preparation + adhesive restoration with CR; G2: (positive control) cavity preparation + GIC restoration; G3: equal to group 1 + CO2 laser with 6 J/cm(2); G4: equal to group 2 + CO2 laser. The blocks were submitted to thermal and pH cycling. Dental demineralization around restorations was quantified using microhardness analyses and Light-Induced Fluorescence (QLF). The groups showed no significant differences in mineral loss at depths between 20 μm and 40 μm. At 60 μm, G2 and G3 ≠ G1, but G4 = G1, G2 and G3. At 80 μm, G4 ≠ G1, and at 100 μm, G4 = G2 = G1. At 140 and 220 μm, G2, G3, and G4 = G1. The averages obtained using QFL in groups 1, 2, 3, and 4 were 0.637, 0.162, 0.095, and 0.048, respectively. QLF and microhardness analyses showed that CO2 laser irradiation reduced mineral loss around the CR restorations but that it did not increase the anticariogenic effect of GIC restorations.
Project description:Preparations of nitrate reductase in the resting state from Pseudomonas aeruginosa exhibit an Mo(V) e.p.r. signal. Progressive reduction of the enzyme results at first in the intensification and then in the disappearance of the signal. Three different species of Mo(V) were detected by e.p.r. These are the high-pH species (g1 = 1.9871; g2 = 1.9795; g3 = 1.9632) and nitrate and nitrite complexes of a low-pH species (respectively g1 = 2.0004; g2 = 1.9858; g3 = 1.9670; and g1 = 1.9975; g2 = 1.9848; g3 = 1.9652). These signals are closely analogous to those for the enzyme from Escherichia coli described by Vincent & Bray [(1978) Biochem. J. 171, 639-647]. Signals typical of iron-sulphur clusters were also detected. In the oxidized enzyme these are believed to arise from a [3Fe-4S] cluster (g = 2.01) and in the reduced enzyme from an unusual low-potential [4Fe-4S]+ cluster (g1 = 2.054; g2 = 1.952; g3 = 1.878). The iron-sulphur centres were also studied in a 'high-catalytic-activity' form of the enzyme. Reduction with Na2S2O4 resulted in the formation of a complex signal with g values at 2.054, 1.952, 1.928, 1.903 and 1.878. The signal could be deconvoluted by reductive titration of the enzyme into two species (g1 = 2.054; g2 = 1.952; g3 = 1.878; and g1 = 2.036; g2 = 1.928; g3 = 1.903). The degradation of a [4Fe-4S] into a [3Fe-4S] cluster in the enzyme is suggested by these studies, the process being dependent on the method used to purify the enzyme. The addition of nitrate to the reduced enzyme results in the oxidation of Mo(IV) to Mo(V) and of all the iron-sulphur centres.