Pyrosequencing Analysis Research Articles

Regulation of adenosine A2A receptor gene expression in a model of binge eating in the amygdaloid complex of female rats.

Pharmacological treatment approaches for eating disorders, such as binge eating disorder and bulimia nervosa, are currently limited. Using a well-characterized animal model of binge eating, we investigated the epigenetic regulation of the A2A Adenosine Receptor (A2AAR) and dopaminergic D2 receptor (D2R) genes. Gene expression analysis revealed a selective increase of both receptor mRNAs in the amygdaloid complex of stressed and restricted rats, which exhibited binge-like eating, when compared to non-stressed and non-restricted rats. Consistently, pyrosequencing analysis revealed a significant reduction of the percentage of DNA methylation but only at the A2AAR promoter region in rats showing binge-like behaviour compared to the control animals. Focusing thus on A2AAR agonist (VT 7) administration (which inhibited the episode of binge systemically at 0.1 mg/kg or intra-central amygdala (CeA) injection at 900 ng/side) induced a significant increase of A2AAR mRNA levels in restricted and stressed rats when compared to the control group. In addition, we observed a significant decrease in A2AAR mRNA levels in rats treated with the A2AAR antagonist (ANR 94) at 1 mg/kg. Consistent changes in the DNA methylation status of the A2AAR promoter were found in restricted and stressed rats after administration of VT 7 or ANR 94. We confirm the role of A2AAR in binge eating behaviours, and we underline the importance of epigenetic regulation of the A2AAR gene, possibly due to a compensatory mechanism to counteract the effect of binge eating. We suggest that A2AAR activation, inducing receptor gene up-regulation, could be relevant to reduction of food consumption.

Interaction among soil physicochemical properties, bacterial community structure, and arsenic contamination: Clay-induced change in long-term arsenic contaminated soils.

Pyrosequencing analyses to determine soil bacterial communities were conducted with forty-two soil samples collected from rice paddy and forest/farmland soils (Group A and B, respectively) at a long-term As-contaminated site. Soil physicochemical properties, such as the concentrations of As, Fe, Al, and Mn, pH, organic matter content, and clay content, were found to be significantly different with land use, and more importantly, strongly affected the bacterial community structure of the soil samples. When fitting the soil properties onto a nonmetric multidimensional scale plot of soil bacterial communities, clay content was found to be the most important factor in clustering the bacterial communities (R2 = 0.4831, p-value = 0.001). Phylum Chloroflexi (-1.03 of bioplot score) and Planctomycetes (1.31 of bioplot score) showed a significant relationship with clay content in soil samples. Interestingly, thebacterial phylotypes linked to clay content were only found in the soil samples of group B with low clay content, and had a strong relationship to As contamination in the redundancy analysis and the correlation analysis.Our results suggest that clay content seems to be negatively related to As contamination in soils, which, in turn, strongly influences the structure of bacterial communities in As-contaminated soil.

Multivariate insights of bulking agents influence on co-biodrying of sewage sludge and food waste: Process performance, organics degradation and microbial community

As a prerequisite additive, bulking agent played an essential role on organic wastes biodrying by affecting the organics degradation and microbial consortia. In this study, a series of experiments were conducted to explore the relationships among the type of bulking agents, organics degradation and microbial community evolution. In line with the excellent physiochemical properties, corncob was found to be more desirable for biodrying with more water removal (62.13% vs. 53.70% for sawdust and 51.72% for straw) and higher energy efficiency. Furthermore, different bulking agents showed different biodegradability and affected co-existed organics degradation. In detail, corncob upgraded the amylase and lipase activities, thus promoting the degradation of readily degradable carbohydrates and lipids in feedstocks, which accounted for >60% of the bio-heat sources for water evaporation. In addition, pyrosequencing analysis revealed that Bacillus (>50%) and Ochrobactrum (>40%) were the dominant genera in thermophilic and cooling phases, with degradation capacities of readily degradable substrate and lignocellulose, respectively. And the pathogens, e.g., E. coli and K. pneumonia, were seriously inhibited by high matrix temperatures in corncob trial. These results not only suggested the corncob was a promising bulking agent, but the potential microbial mechanisms for organics degradation were also revealed.

ZNF492 and GPR149 methylation patterns as prognostic markers for clear cell renal cell carcinoma: Array‑based DNA methylation profiling.

The present study aimed to identify novel methylation markers of clear cell renal cell carcinoma (ccRCC) using microarray methylation analysis and evaluate their prognostic relevance in patient samples. To identify cancer‑specific methylated biomarkers, microarray profiling of ccRCC samples from our institute (n=12) and The Cancer Genome Atlas (TCGA) database (n=160) were utilized, and the prognostic relevance of candidate genes were investigated in another TCGA dataset (n=153). For validation, pyrosequencing analyses with ccRCC samples from our institute (n=164) and another (n=117) were performed and the potential clinical application of selected biomarkers was examined. We identified 22 CpG island loci that were commonly hypermethylated in ccRCC. Kaplan‑Meier analysis of TCGA data indicated that only 4/22 loci were significantly associated with disease progression. In the internal validation set, Kaplan‑Meier analysis revealed that hypermethylation of two loci, zinc finger protein 492 (ZNF492) and G protein‑coupled receptor 149 (GPR149), was significantly associated with shorter time‑to‑progression. Multivariate Cox regression models revealed that hypermethylation of ZNF492 [hazard ratio (HR), 5.44; P=0.001] and GPR149 (HR, 7.07; P<0.001) may be independent predictors of tumor progression. Similarly, the methylation status of these two genes was significantly associated with poor outcomes in the independent external validation cohort. Collectively, the present study proposed that the novel methylation markers ZNF492 and GPR149 could be independent prognostic indicators in patients with ccRCC.

Enhancing electricity generation of microbial fuel cell for wastewater treatment using nitrogen-doped carbon dots-supported carbon paper anode

Microbial fuel cell is a promising technology to harvest energy from wastewater, and effective anode catalysts are essential for its applications. In this study, zero-dimension nitrogen-doped carbon dots, as biocompatible efficient modifiers, were synthesized and decorated onto the carbon paper surface to prepare microbial fuel cell anode. The as-fabricated nitrogen-doped carbon dots decorated carbon paper anode was able to enhance electricity generation and efficient wastewater treatment. It was found that the as-fabricated electrode had a high hydrophilicity, which was favorable for the microbial immobilization. Compared with the carbon paper anode, the biofilm on the as-prepared anode was almost twice of thickness. After introducing nitrogen-doped carbon dots, the extracellular electron transfer process from the microorganisms to the anode was enhanced. The microbial fuel cell with the as-developed anode generated a remarkable power output of 0.32 mW, 1.1 times higher than that of the raw carbon paper anode with same measurement area. Moreover, 16 sRNA pyrosequencing analysis shows that the quantity of Pseudomonas on the electrode increased by ∼40%, which also promoted the electron transfer rate by excreting mediators like phenazines or phenazine derivatives. This work developed a new approach to design microbial fuel cell anode material and fabricate a high-efficiency and environmentally friendly anode for electricity generation from wastewater.

Jointly reducing antibiotic resistance genes and improving methane yield in anaerobic digestion of chicken manure by feedstock microwave pretreatment and activated carbon supplementation

Antibiotic resistance genes (ARGs) stemming from animal manure pose adverse impacts on human health and ecology and consequently cost more to treat the anaerobic digestate after manure fermentation. To jointly reduce the ARGs and improve the methane yield during anaerobic digestion (AD) of chicken manure, microwave pretreatment of manure and activated carbon supplementation were simultaneously applied and evaluated in an anaerobic digester (R1). For comparison, three other digesters, a digester (R2) fed with raw chicken manure without activated carbon, a digester (M1) fed with microwave-pretreated chicken manure and without activated carbon, and a digester (M2) fed with raw chicken manure supplemented with activated carbon, were operated in parallel. The results showed that after 47 days of AD operation, R1 achieved an 87–95% ARGs removal rate, significantly higher than that of R2 (34–58%). As the loading rate was 5.8–7.0 g volatile solids (VS)/L, the average methane yield of R1 was 0.287 L/g VS, which was 1.4-fold, 1.3-fold and 1.1-fold higher than that of R2, M1 and M2, respectively. The increased methane yield was ascribed to the combined effect of feedstock microwave pretreatment and activated carbon supplementation. Pyrosequencing analysis showed that the dominant bacterial phyla were Firmicutes, Bacteroidetes and Proteobacteria while the dominant archaeal genus were Methanosarcina, Methanothrix, Methanolinea, Methanomassiliicoccus, Methanospirillum, Methanoregula and Methanobacterium. The KEGG cluster analysis and functional analysis of genome from archaeal communities demonstrated that the dominant functions enhanced by substrate microwave pretreatment and activated carbon supplementation were linked to amino acid metabolism, carbohydrate metabolism, cell communication, energy metabolism, signaling molecules and interaction, and signal transduction.

160 Mechanisms governing promoter methylation and apoptosis in cutaneous T-cell lymphoma: implications for therapy with methotrexate and JAK-inhibitors

Cutaneous T-cell lymphoma (CTCL) is characterized by resistance to apoptosis involving dysregulation of the FAS ligand (FASL) - FAS death receptor pathway. CTCL cells also exhibit constitutively activated STAT proteins, which are known to upregulate and bind DNA methyltransferases (DNMTs). We hypothesized that, like many other tumor suppressor genes, FAS and FASL expression is at least partially regulated by promoter methylation. Earlier, we showed that methotrexate (MTX), a known folate antagonist, acts as a DNA methylation inhibitor via depleting the methyl donor, S-adenosylmethionine (SAM), in CTCL cells. Pyrosequencing analysis of FAS and FASL promoters showed that MTX inhibited methylation of certain CpG sites in these genes, which resulted in increased FAS/FASL expression and subsequent apoptosis via activated caspase 8. We found that shRNA mediated genetic knockdown of DNMT -1 and -3A also resulted in upregulation of FASL mRNA and protein in CTCL cell lines. However, the reduction in FASL promoter methylation induced by MTX was generally superior to that induced by shRNA knockdown and was not improved by a combination of MTX plus knockdown. Because STAT proteins help regulate DNMTs, we also evaluated the effect of multiple JAK inhibitors (fedratinib, tofacitinib, and ruxolitinib) in CTCL cells and Sezary patient’s blood. Among these, fedratinib treatment was most effective in decreasing viability, spheroid formation, and inducing apoptosis. These responses were accompanied by decreased phosphorylation of STAT3 at tyrosine-705. Taken together, our data demonstrate that enhanced apoptosis of CTCL can be induced by MTX, DNMT knockdown and JAK inhibitors. This is consistent with a mechanistic cascade in which activated STATs enhance DNMT activities that in turn suppress the expression of apoptotic factors and promote CTCL survival. Our findings suggest a therapeutic role for MTX and JAK inhibitors in the management of CTCL.

Epigenetically altered miR‑193a‑3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7.

Emerging evidence has demonstrated that microRNAs (miRNAs/miRs) have various biological functions in the development of human epidermal growth factor receptor 2 (HER2) positive breast cancer. The aim of the present study is to reveal the mechanism of miR-193a-3p inhibiting the progress of HER2 positive breast cancer. The expression of miR-193a-3p was evaluated by quantitative polymerase chain reaction (PCR). The methylation status of miR-193a-3p was evaluated by PCR and pyrosequencing analysis. Overexpression of miR-193a-3p and growth factor receptor bound protein 7 (GRB7) combined with in vitro tumorigenic assays were conducted to determine the carcinostatic capacities of miR-193a-3p in HER2 positive breast cancer cells. The association between miR-193a-3p and GRB7 was determined by luciferase reporter assay. Protein level was evaluated using western blot analysis. miR-193a-3p was down-regulated in HER2 positive breast cancer cells and clinical tissues. Methylation-mediated silencing led to decreased expression of miR-193a-3p in HER2 positive breast cancer. Overexpression of miR-193a-3p could inhibit proliferation, migration and invasion of breast cancer cells. Overexpression of GRB7 could abolish this effect. miR-193a-3p could directly target the 3′ untranslated region of GRB7. miR-193a-3p could directly or indirectly target extracellular signal-regulated kinase 1/2 (ERK1/2) and forkhead box M1 (FOXM1) signaling. In conclusion, it was identified that silencing of miR-193a-3p through hypermethylation can promote HER2 positive breast cancer progress by targeting GRB7, ERK1/2 and FOXM1 signaling. The function of miR-193a-3p in HER2 positive breast cancer implicates its potential application in therapy.

Gut microbiota in obstructive sleep apnea-hypopnea syndrome: disease-related dysbiosis and metabolic comorbidities.

Gut microbiota alterations manifest as intermittent hypoxia and fragmented sleep, thereby mimicking obstructive sleep apnea–hypopnea syndrome (OSAHS). Here, we sought to perform the first direct survey of gut microbial dysbiosis over a range of apnea–hypopnea indices (AHI) among patients with OSAHS. We obtained fecal samples from 93 patients with OSAHS [5 < AHI ≤ 15 (n=40), 15 < AHI ≤ 30 (n=23), and AHI ≥ 30 (n=30)] and 20 controls (AHI ≤ 5) and determined the microbiome composition via 16S rRNA pyrosequencing and bioinformatics analysis of variable regions 3–4. We measured fasting levels of homocysteine (HCY), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). Results revealed gut microbial dysbiosis in several patients with varying severities of OSAHS, reliably separating them from controls with a receiver operating characteristic-area under the curve (ROC-AUC) of 0.789. Functional analysis in the microbiomes of patients revealed alterations; additionally, decreased in short-chain fatty acid (SCFA)-producing bacteria and increased pathogens, accompanied by elevated levels of IL-6. Lactobacillus levels correlated with HCY levels. Stratification analysis revealed that the Ruminococcus enterotype posed the highest risk for patients with OSAHS. Our results show that the presence of an altered microbiome is associated with HCY among OSAHS patients. These changes in the levels of SCFA affect the levels of pathogens that play a pathophysiological role in OSAHS and related metabolic comorbidities.

Analysis of oral microbiota in patients with obstructive sleep apnea-associated hypertension

Obstructive sleep apnea–hypopnea syndrome (OSAHS) is an independent risk factor for hypertension (HTN). The oral microbiota plays a pathophysiological role in cardiovascular diseases; however, there are few reports directly investigating and identifying the organisms involved in OSAHS-related HTN. Therefore, this study aimed to identify those organisms. We obtained 139 oral samples and determined the microbiome composition using pyrosequencing and bioinformatic analyses of the 16S rRNA. We examined the fasting levels of cytokines and homocysteine in all participants and analyzed the correlations between the oral microbiota and homocysteine levels. We determined the molecular mechanism underlying HTN by investigating the genetic composition of the strains in the blood. We detected higher relative abundances of Porphyromonas and Aggregatibacter and elevated proinflammatory cytokines in patients with OSAHS of varying severity compared with individuals without OSAHS; however, the two organisms were not measured in the blood samples from all participants. High levels of specific Porphyromonas bacteria were detected in patients with OSAHS with and without HTN, whereas the relative abundance of Aggregatibacter was negatively correlated with the homocysteine level. The receiver operating characteristic curve analysis of controls and patients with OSAHS resulted in area under the curve values of 0.759 and 0.641 for patients with OSAHS with or without HTN, respectively. We found that the predictive function of oral microbiota was different in patients with OSAHS with and without HTN. However, there was no direct invasion by the two organisms causing endothelial cell injury, leading to speculation regarding the other mechanisms that may lead to HTN. Elucidating the differences in the oral microbiome will help us understand the pathogenesis of OSAHS-related HTN.

Urban traffic changes the biodiversity, abundance, and activity of phyllospheric nitrogen-fixing bacteria.

The phyllosphere provides appropriate conditions for colonization by microorganisms, including diazotrophic bacteria. However, a poor understanding of the effects of the atmospheric environment on the phyllospheric diazotrophic communities persists. We detected the biodiversity, abundance, and activity of nitrogen-fixing bacteria in the phyllospheres of two evergreen shrubs, Nerium indicum Mill. and Osmanthus sp., sampled from urban areas with heavy traffic, a college campus, and a forest. Quantitative PCR analysis indicated that the copy numbers of nifH sequences were highest in the phyllospheres of both plants in heavy-traffic urban areas and correlated with the recorded nitrogenase activities of the phyllospheres. Similarly, the phyllosphere from heavy-traffic urban areas also possessed the highest biodiversity indices of diazotrophic communities from both the two plants. Pyrosequencing analysis revealed a diversity of nifH sequences in phyllosphere that were mostly uniquely found in the phyllosphere, and many of these were proteobacteria-like and cyanobacteria-like. Members of the Proteobacteria, mostly of which were not closely related to unknown organisms, were detected exclusively in the phyllosphere and represented substantial fractions of their associated diazotrophic communities. Our study provides initial insight into the shifts in the biodiversity and community structure of N2-fixing microorganisms in the phyllospheres of different atmospheric environments.

Disruption of sleep architecture in Prevotella enterotype of patients with obstructive sleep apnea-hypopnea syndrome.

IntroductionIntermittent hypoxia and sleep fragmentation are critical pathophysiological processes involved in obstructive sleep apnea‐hypopnea syndrome (OSAHS). Those manifestations independently affect similar brain regions and contribute to OSAHS‐related comorbidities that are known to be related to the host gut alteration microbiota. We hypothesized that gut microbiota disruption may cross talk the brain function via the microbiota–gut–brain axis. Thus, we aim to survey enterotypes and polysomnographic data of patients with OSAHS.MethodsSubjects were diagnosed by polysomnography, from whom fecal samples were obtained and analyzed for the microbiome composition by variable regions 3–4 of 16S rRNA pyrosequencing and bioinformatic analyses. We examined the fasting levels of interleukin‐6 and tumor necrosis factor‐alpha of all subjects.ResultsThree enterotypes Bacteroides, Ruminococcus, and Prevotella were identified in patients with OSAHS. Arousal‐related parameters or sleep stages are significantly disrupted in apnea‐hypopnea index (AHI) ≥15 patients with Prevotella enterotype; further analysis this enterotype subjects, obstructive, central, and mixed apnea indices, and mean heart rate are also significantly elevated in AHI ≥15 patients. However, blood cytokines levels of all subjects were not significantly different.ConclusionsThis study indicates the possibility of pathophysiological interplay between enterotypes and sleeps structure disruption in sleep apnea through a microbiota–gut–brain axis and offers some new insight toward the pathogenesis of OSAHS.

Nonadditive and allele-specific expression of insulin-like growth factor 1 in Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (O. aureus, ♂) hybrids.

Hybrid Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (O. aureus, ♂) is a widely cultured tilapia variety due to its growth vigor compared to the parent species. As a peptide hormone, insulin-like growth factor 1 (IGF-1) plays a critical role in regulating somatic growth. The present study focuses on the expression characteristics of IGF-1 in hybrid tilapia. The cloned complete open reading frame of IGF-1 in hybrid tilapia is 549 bp in length, encoding a protein of 182 amino acids. The deduced protein is highly similar to that of Nile tilapia and blue tilapia. IGF-1 was found to be primarily expressed in the liver and muscle in the hybrid; lower expression levels were found in other tissues such as the intestine, spleen, and head-kidney. Increased mRNA expression was observed in the liver and muscle of the hybrid compared to Nile tilapia and blue tilapia, indicating a nonadditive expression pattern in the hybrid. An IGF-1 SNP site (397 site: C in Nile tilapia, G in blue tilapia) for differentiating the Nile tilapia or blue tilapia subgenome in hybrids was identified. Pyrosequencing analysis of the liver transcriptome indicated that most of the hybrids (9 of 10 individuals) predominantly expressed the G allele, demonstrating bias of the blue tilapia subgenome. The present study provides novel data indicating, for the first time, overall gene expression of IGF-1 and allele-specific expression in hybrid tilapia.

Biological oxidation of choline‐based ionic liquids in sequencing batch reactors

AbstractBACKGROUNDIonic liquids (ILs) are salts consisting of an organic cation and an organic or inorganic anion that are potential use for organic synthesis, biomass conversion, gas absorption, polymerization, metal extraction and lubrication. We examined the biodegradability of choline chloride (CholineCl), choline acetate (CholineAc) and choline bis(trifluoromethylsulfonyl)imide (CholineNTf2) in a sequencing batch reactor (SBR) to assess the influence of the IL anion.RESULTSFollowing acclimation, activated sludge exhibited good activity, and high COD (80–90%) and TOC removal (75–85%) with initial concentrations of CholineCl and CholineAc over the range 0.25–15 mmol L–1. However, increased concentrations of CholineNTf2 in the reactor feed had an inhibitory effect on the sludge. An analysis of the reaction effluent revealed the presence of the main intermediates of choline degradation: ethanol, and methyl‐, dimethyl‐ and trimethylamine. The initial and final microbial communities of the sludge were characterized by pyrosequencing analysis, and the latter was found to consist mainly of Alfa‐, Beta‐ and Gamma‐proteobacteria.CONCLUSIONCholine‐based ionic liquids can be efficiently removed in SBRs. Complete choline degradation (0.25–15 mmol L–1) was accomplished at a variable depletion rate depending on the particular IL anion (chloride, acetate or NTf2). © 2019 Society of Chemical Industry

Effects of a polysaccharide-rich extract derived from Irish-sourced Laminaria digitata on the composition and metabolic activity of the human gut microbiota using an in vitro colonic model

BackgroundBrown seaweeds are known to be a rich source of fiber with the presence of several non-digestible polysaccharides including laminarin, fucoidan and alginate. These individual polysaccharides have previously been shown to favorably alter the gut microbiota composition and activity albeit the effect of the collective brown seaweed fiber component on the microbiota remains to be determined.MethodsThis study investigated the effect of a crude polysaccharide-rich extract obtained from Laminaria digitata (CE) and a depolymerized CE extract (DE) on the gut microbiota composition and metabolism using an in vitro fecal batch culture model though metagenomic compositional analysis using 16S rRNA FLX amplicon pyrosequencing and short-chain fatty acid (SCFA) analysis using GC-FID.ResultsSelective culture analysis showed no significant changes in cultured lactobacilli or bifidobacteria between the CE or DE and the cellulose-negative control at any time point measured (0, 5, 10, 24, 36, 48 h). Following metagenomic analysis, the CE and DE significantly altered the relative abundance of several families including Lachnospiraceae and genera including Streptococcus, Ruminococcus and Parabacteroides of human fecal bacterial populations in comparison to cellulose after 24 h. The concentrations of acetic acid, propionic acid, butyric acid and total SCFA were significantly higher for both the CE and DE compared to cellulose after 10, 24, 36 and 48 h fermentation (p < 0.05). Furthermore, the acetate:propionate ratio was significantly reduced (p < 0.05) for both CD and DE following 24, 36 and 48 h fermentation.ConclusionThe microbiota-associated metabolic and compositional changes noted provide initial indication of putative beneficial health benefits of L. digitata in vitro; however, research is needed to clarify if L. digitata-derived fiber can favorably alter the gut microbiota and confer health benefits in vivo.

Effect of the Lactobacillus rhamnosus strain GG and tagatose as a synbiotic combination in a dextran sulfate sodium-induced colitis murine model

Synbiotics, a combination of prebiotics and probiotics, produce synergistic effects to promote gastrointestinal health. Herein, we investigated the synbiotic interaction between the Lactobacillus rhamnosus strain GG (LGG; a probiotic strain) and tagatose (a prebiotic) in a dextran sulfate sodium (DSS)-induced colitis murine model. Initially, body weight, food intake, and clinical features were dramatically decreased after treatment with DSS, and the addition of LGG, tagatose, or both ameliorated these effects. In our pyrosequencing analysis of fecal microbiota, DSS treatment increased the abundance of Proteobacteria and decreased that of Firmicutes. When LGG and tagatose were administered as synbiotics, the gut microbiota composition recovered from the dysbiosis caused by DSS treatment. In particular, the abundance of Bacteroides, Lactobacillus, and Akkermansia was significantly associated with probiotic, prebiotic, and synbiotic treatments. Taken together, our results suggest that LGG and tagatose as synbiotics can alleviate colitis, and synbiotics could be applied as dietary supplements in dairy foods such as yogurt and cheese.

Enrichment of bacteria and alginate lyase genes potentially involved in brown alga degradation in the gut of marine gastropods

Gut bacteria of phytophagous and omnivorous marine invertebrates often possess alginate lyases (ALGs), which are key enzymes for utilizing macroalgae as carbon neutral biomass. We hypothesized that the exclusive feeding of a target alga to marine invertebrates would shift the gut bacterial diversity suitable for degrading the algal components. To test this hypothesis, we reared sea hare (Dolabella auricularia) and sea snail (Batillus cornutus) for two to four weeks with exclusive feeding of a brown alga (Ecklonia cava). Pyrosequencing analysis of the gut bacterial 16S rRNA genes revealed shifts in the gut microbiota after rearing, mainly due to a decrease in the variety of bacterial members. Significant increases in six and four 16S rRNA gene phylotypes were observed in the reared sea hares and sea snails, respectively, and some of them were phylogenetically close to known alginate-degrading bacteria. Clone library analysis of PL7 family ALG genes using newly designed degenerate primer sets detected a total of 50 ALG gene phylotypes based on 90% amino acid identity. The number of ALG gene phylotypes increased in the reared sea hare but decreased in reared sea snail samples, and no phylotype was shared between them. Out of the 50 phylotypes, 15 were detected only after the feeding procedure. Thus, controlled feeding strategy may be valid and useful for the efficient screening of genes suitable for target alga fermentation.

Haemolymph microbiome of the cultured spiny lobster Panulirus ornatus at different temperatures

Lobsters have an open circulatory system with haemolymph that contains microorganisms even in the healthy individuals. Understanding the role of these microorganisms becomes increasingly important particularly for the diagnosis of disease as the closed life-cycle aquaculture of the spiny lobster Panulirus ornatus nears commercial reality. This study aimed to characterise haemolymph responses of healthy cultured P. ornatus juveniles at control (28 °C) and elevated (34 °C) temperatures. This was assessed by measuring immune parameters (total granulocyte counts, total haemocyte counts, clotting times), and culture-independent (pyrosequencing of haemolymph DNA) and culture-dependent (isolation using nonselective growth medium) techniques to analyse bacterial communities from lobster haemolymph sampled on days 0, 4 and 6 post-exposure to the temperature regimes. Elevated temperature (34 °C) affected lobster survival, total granulocyte counts, and diversity, load and functional potential of the haemolymph bacterial community. Pyrosequencing analyses showed that the core haemolymph microbiome consisted of phyla Proteobacteria and Bacteriodetes. Overall, culture-independent methods captured a higher bacterial diversity and load when compared to culture-dependent methods, however members of the Rhodobacteraceae were strongly represented in both analyses. This is the first comprehensive study providing comparisons of haemolymph bacterial communities from healthy and thermally stressed cultured juvenile P. ornatus and has the potential to be used in health monitoring programs.

Enhanced Degradation of Waste Activated Sludge in Microbial Electrolysis Cell by Ultrasonic Treatment.

This study investigates the feasibility of ultrasonic pretreatment for improving treatment efficiency of waste activated sludge (WAS) in microbial electrolysis cell (MEC). Results showed that at applied voltage of 0.5 V, biogas production and cathodic hydrogen recovery enhanced 3.68-fold and 2.56-fold, respectively. Due to the transformation of soluble COD accelerated by the pretreatment, the removal rates of suspended solids and volatile suspended solids were significantly enhanced by 1.38-fold and 1.48-fold, respectively. Various kinds of organics, including VFAs (volatile fatty acids), proteins and carbohydrates, could be utilized in sequence. The primary biodegradable substance in MEC was hydrophilic fraction from sludge organics and the pretreatment effectively resulted in an elevated concentration of this fraction. The 16S rRNA pyrosequencing analysis demonstrated multiple syntrophic interactions between fermentative bacteria, exoelectrogenes, and methanogenic archaea in MEC for WAS.

Improved lignin degradation through distinct microbial community in subsurface sediments of one eutrophic lake

Lignocellulose is a promising sustainable alternative fuel and its decomposition is critical for the formation of sediment organic matter. The lignin of lignocellulose limits the access of enzymes to cellulose and slows down the decomposition. Therefore, the development of efficient microbial consortia to deconstruct lignin is of great interest. In this study, a 715-day in situ experiment was performed to investigate the degradation process of macrophyte Phragmites australis debris at different depths of sediments (up to 38 cm) in a eutrophic lake. Although litter mass degradation showed an obvious decreasing tendency as the depth increased, the efficiency of lignin degradation in subsurface sediments was significance higher than that in surface sediments (p < 0.05). Pyrosequencing analysis showed that the subsurface sediments contained the specific anaerobic bacteria (genus Sulfuricurvum, Clostridium and Treponema) and anaerobic fungi (phylum Chytridiomycota and Rozellomycota). These anaerobic microbes well adapted to the degradation of recalcitrant organic matter, and led to the increasing activity of lignin biodegradation in subsurface sediments. This work could help improve our understanding of lignin biodegradation and develop a potential biotechnology for efficient biofuel production.