Mechanisms underlying Clostridium pasteurianum's metabolic shift when grown with Geobacter sulfurreducens.

Recently, a study showed that glycerol fermentation by Clostridium pasteurianum could be metabolically redirected when the electroactive bacterium Geobacter sulfurreducens was added in the culture. It was assumed that this metabolic shift of the fermentative species resulted from an interspecies electron transfer. The aim of this study was to find out the mechanisms used for this interaction and how they affect the metabolism of C. pasteurianum. To get insights into the mechanisms involved, several coculture setups and RNA sequencing with differential expression analysis were performed. As a result, a putative interaction model was proposed: G. sulfurreducens produces cobamide molecules that possibly modify C. pasteurianum metabolic pathway at the key enzyme glycerol dehydratase, and affect its vanadium nitrogenase expression. In addition, the results suggested that G. sulfurreducens' electrons could enter C. pasteurianum through its transmembrane flavin-bound polyferredoxin and cellular cytochrome b5-rubredoxin interplay, putatively reinforcing the metabolic shift. Unravelling the mechanisms behind the interaction between fermentative and electroactive bacteria helps to better understand the role of bacterial interactions in fermentation setups. KEY POINTS: • C. pasteurianum-G. sulfurreducens interaction inducing a metabolic shift is mediated • C. pasteurianum's metabolic shift in coculture might be induced by cobamides • Electrons possibly enter C. pasteurianum through a multiflavin polyferredoxin.

JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation.

Hyperosmotic Stress Induces Tau Proteolysis by Caspase-3 Activation in SH-SY5Y Cells.

Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of µ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor µ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.

Influence of process dynamics on the microbial diversity in a nitrifying biofilm reactor: Correlation analysis and simulation study.

For engineers, it is interesting to gain insight in the effect of control strategies on microbial communities, on their turn influencing the process behavior and its stability. This contribution assesses the influence of process dynamics on the microbial community in a biofilm reactor for nitrogen removal, which was controlled according to several strategies aiming at nitrite accumulation. The process dataset, combining conventional chemical and physical data with molecular information, was analyzed through a correlation analysis and in a simulation study. During nitrate formation, an increased nitrogen loading rate (NLR) resulted in a drop of the bulk liquid oxygen concentration without resulting in nitrite accumulation. A biofilm model was able to reproduce the bulk liquid nitrogen concentrations in two periods before and after this increased NLR. As the microbial parameters calibrated for the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in both periods were different, it was concluded that the increased NLR governed an AOB and NOB population shift. Based on the molecular data, it was assumed that each period was typified by one dominant AOB and probably several subdominant NOB populations. The control strategies for nitrite accumulation influenced the bulk liquid composition by controlling the competition between AOB and NOB. Biotechnol. Bioeng. 2016;113: 1962-1974. © 2016 Wiley Periodicals, Inc.

Biomass acid pretreatment impacts on metabolic routes and bacterial composition of dark fermentation process.

Complex organic matter represents a suitable substrate to produce hydrogen through dark fermentation (DF) process. To increase H2 yields, pretreatment technology is often required. The main objective of the present work was to investigate thermo-acid pretreatment impact on sugar solubilization and biotic parameters of DF of sorghum or organic fraction of municipal solid waste (OFMSW). Biochemical hydrogen potential tests were carried out without inoculum using raw or thermo-acid pretreated substrates. Results showed an improvement in sugar solubilization after thermo-acid pretreatments. Pretreatments led to similar DF performances (H2 and total metabolite production) compared to raw biomasses. Nevertheless, they were responsible for bacterial shifts from Enterobacteriales towards Clostridiales and Bacillales as well as metabolic changes from acetate towards butyrate or ethanol. The metabolic changes were attributed to the biomass pretreatment impact on indigenous bacteria as no change in the metabolic profile was observed after performing thermo-acid pretreatments on irradiated OFMSW (inactivated indigenous bacteria and inoculum addition). Consequently, acid pretreatments were inefficient to improve DF performances but led to metabolic and bacterial community changes due to their impact on indigenous bacteria.

Role of endogenous soil microorganisms in controlling antimicrobial resistance after the exposure to treated wastewater.

The reuse of treated wastewater (TWW) for irrigation appears to be a relevant solution to the challenges of growing water demand and scarcity. However, TWW contains not only micro-pollutants including pharmaceutical residues but also antibiotic resistant bacteria. The reuse of TWW could contribute to the dissemination of antimicrobial resistance in the environment. The purpose of this study was to assess if exogenous bacteria from irrigation waters (TWW or tap water-TP) affect endogenous soil microbial communities (from 2 soils with distinct irrigation history) and key antibiotic resistance gene sul1 and mobile genetic elements intl1 and IS613. Experiments were conducted in microcosms, irrigated in one-shot, and monitored for three months. Results showed that TP or TWW exposure induced a dynamic response of soil microbial communities but with no significant increase of resistance and mobile gene abundances. However, no significant differences were observed between the two water types in the current experimental design. Despite this, the 16S rDNA analysis of the two soils irrigated for two years either with tap water or TWW resulted in soil microbial community differentiation and the identification of biomarkers from Xanthomonadaceae and Planctomycetes families for soils irrigated with TWW. Low-diversity soils were more sensitive to the addition of TWW. Indeed, TWW exposure stimulated the growth of bacterial genera known to be pathogenic, correlating with a sharp increase in the copy number of selected resistance genes (up to 3 logs). These low-diversity soils could thus enable the establishment of exogenous bacteria from TWW which was not observed with native soils. In particular, the emergence of Planctomyces, previously suggested as a biomarker of soil irrigated by TWW, was here demonstrated. Finally, this study showed that water input frequency, initial soil microbial diversity and soil history drive changes within soil endogenous communities and the antibiotic resistance gene pool.

Conditions for efficient alkaline storage of cover crops for biomethane production.

An innovative process aiming to combine storage and alkali pretreatment of cover crops was investigated using lime as a low cost and environmental friendly reactant. Different lime loadings and Total Solid concentrations (TS) allowed to highlight the abiotic mechanisms of deacetylation during the early stages of the process. Long-term storage experiments of rye and sunflower cover crops at 100 g.kgTS-1 lime loading allowed to evaluate the fermentation kinetics and to compare performances in dry and wet conditions to classical silage storage. The dry condition allowed an efficient alkaline storage and up to a 15.7% Biochemical Methane Potential (BMP) increase, while the wet condition underwent a succession of fermentations with a high butyric acid accumulation and H2 production, leading to a 13% BMP loss. Silage experiments allowed an efficient preservation of the BMP, with no significant variation over the 6-month storage duration.

Improving the storage of cover crops by co-ensiling with different waste types: Effect on fermentation and effluent production.

Cover crops harvested at a low maturity stage generally have a high moisture content, which may generate energy losses during silage storage via effluent production and undesirable fermentations. This paper investigates the use of different waste types as absorbent co-substrates to be added before ensiling. The relation between the absorbent water holding capacity and silage effluent volume was first studied to find an effective parameter to prevent effluent production. Effluent retention was found to be proportional to the absorbent loading and water holding capacity (r2 = 0.98) and up to 90 % of effluent production was avoided when compared to control (295 l.t-1). The impact of different co-substrates (including bio-waste and manures) on overall ensiling performances was then investigated at an optimized absorbent loading. All co-substrates allowed a total effluent retention while a 76 l.t-1 effluent volume was reported for the control. The silage fermentation was modified or mostly unchanged depending on the co-substrate chemical and microbial properties and different metabolic pathways were observed (e.g. homolactic or butyric fermentation). In most conditions, the methane potential of the crop was efficiently preserved over a storage of 60 days. Co-ensiling was shown to be a relevant silage preparation method for biogas production.

Microbial community redundance in biomethanation systems lead to faster recovery of methane production rates after starvation.

The Power-to-Gas concept corresponds to the use of the electric energy surplus to produce H2 by water electrolysis, that can be further converted to methane by biomethanation. However, the fluctuant production of renewable energy sources can lead to discontinuous H2 injections into the reactors, that may interfere with the adaptation of the microbial community to high H2 partial pressures. In this study, the response of the microbial community to H2 and organic feed starvation was evaluated in in-situ and ex-situ biomethanation. The fed-batch reactors were fed with acetate or glucose and H2, and one or four weeks of starvation periods were investigated. Methane productivity was mostly affected by the four-week starvation period. However, both in-situ and ex-situ biomethanation reactors recovered their methane production rate after starvation within approximately one-week of normal operation, while the anaerobic digestion (AD) reactors did not recover their performances even after 3 weeks of normal operation. The recovery failure of the AD reactors was probably related to a slow growth of the syntrophic and methanogen microorganisms, that led to a VFA accumulation. On the contrary, the faster recovery of both biomethanation reactors was related to the replacement of Methanoculleus sp. by Methanobacterium sp., restoring the methane production in the in-situ and ex-situ biomethanation reactors. This study has shown that biomethanation processes can respond favourably to the intermittent H2 addition without compromising their CH4 production performance.

Etiology of hepatocellular carcinoma in Latin America: a prospective, multicenter, international study.

BACKGROUND/AIMS: No prospective study has been published investigating etiology of HCC in Latin America. The primary aim of this prospective study was to analyze the etiology of liver disease in patients with HCC from our area. Secondary aims were to evaluate staging using Okuda and BCLC classifications; and percentage of patients receiving treatment. METHODS: The Governing Board of the Latin American Association for the Study of the Liver designed the protocol. During a 18 month period, all members were invited to load their incident HCC cases on line. RESULTS: 240 cases from 9 countries were uploaded, 174 were male (72.5%), median age was 64 years, interquartile range 57-72. In 85.4% of cases, patients had underlying cirrhosis. Main etiological factors were: HCV in 74 patients (30.8%), alcohol in 49 (20.4%), cryptogenic cirrhosis in 35 (14.6%), HBV in 26 (10.8%), HCV plus alcohol in 14 (5.8%). Considering the combinations, hepatitis C was shown in 91 patients (38%); chronic alcoholism in 68 patients (28%); and hepatitis B in 33 patients (14%). There were no significant differences between the groups in the age at diagnosis. Percentage of male gender was higher in groups of alcohol (94%), HCV plus alcohol (93%) and HBV (85%) than in cryptogenic cirrhosis (60%) and HCV (59%) (p<0.001). CONCLUSIONS: Our prospective study showed that hepatitis C is the more frequent etiology of HCC in Latin America, followed by alcoholic cirrhosis. Demographical results showed a male predominance (male:female ratio 2.6) with an important proportion of patients being diagnosed at their sixties.

Role of indigenous bacteria in dark fermentation of organic substrates.

Hydrogen production by dark fermentation of complex organic substrates, such as biowaste, can naturally take place with indigenous bacteria or by adding an external microbial inoculum issued from various natural environments. This study aims to determine whether indigenous bacteria associated with thermal pretreatment could impact dark fermentation performances. Biochemical hydrogen potential tests were carried out on seven organic substrates. Results showed a strong influence of the indigenous bacteria which are as effective as thermally pretreated exogenous bacteria to produce H2 and metabolites. High abundance in Clostridiales and/or Enterobacteriales was associated with high H2 yield. This study shows that no inoculum nor pretreatment are required to achieve satisfactory dark fermentation performances from organic waste.

Biomethanation processes: new insights on the effect of a high H2 partial pressure on microbial communities.

BACKGROUND: Biomethanation is a promising solution to upgrade the CH4 content in biogas. This process consists in the injection of H2 into an anaerobic digester, using the capacity of indigenous hydrogenotrophic methanogens for converting the injected H2 and the CO2 generated from the anaerobic digestion process into CH4. However, the injection of H2 could cause process disturbances by impacting the microbial communities of the anaerobic digester. Better understanding on how the indigenous microbial community can adapt to high H2 partial pressures is therefore required. RESULTS: Seven microbial inocula issued from industrial bioprocesses treating different types of waste were exposed to a high H2 partial pressure in semi-continuous reactors. After 12 days of operation, even though both CH4 and volatile fatty acids (VFA) were produced as end products, one of them was the main product. Acetate was the most abundant VFA, representing up to 94% of the total VFA production. VFA accumulation strongly anti-correlated with CH4 production according to the source of inoculum. Three clusters of inocula were distinguished: (1) inocula leading to CH4 production, (2) inocula leading to the production of methane and VFA in a low proportion, and (3) inocula leading to the accumulation of mostly VFA, mainly acetate. Interestingly, VFA accumulation was highly correlated to a low proportion of archaea in the inocula, a higher amount of homoacetogens than hydrogenotrophic methanogens and, the absence or the very low abundance in members from the Methanosarcinales order. The best methanogenic performances were obtained when hydrogenotrophic methanogens and Methanosarcina sp. co-dominated all along the operation. CONCLUSIONS: New insights on the microbial community response to high H2 partial pressure are provided in this work. H2 injection in semi-continuous reactors showed a significant impact on microbial communities and their associated metabolic patterns. Hydrogenotrophic methanogens, Methanobacterium sp. or Methanoculleus sp. were highly selected in the reactors, but the presence of co-dominant Methanosarcinales related species were required to produce higher amounts of CH4 than VFA.

Effect of ammonium, electron donor and sulphate transient feeding conditions on sulphidogenesis in sequencing batch bioreactors.

This work aimed to study the effect of transient feeding conditions on sulphidogenesis in 8 sequencing batch bioreactors (SBR). SBR L1 and H1, operated under steady-state conditions were used as the control reactors, while four SBR were tested under transient feeding conditions using moderate (L2 and L3, feast and famine: 2.5 and 0 g SO42-·L-1) and high (H2 and H3, feast and famine: 15 and 0 g SO42-·L-1) loads. The sulphate removal efficiency (RE) was ≥90% in SBR L2, L3 and H1. The NH4+ famine conditions resulted in a higher sulphate RE (≥40% H3) compared to feast conditions (≤20% H2). Besides, the sulphidogenic first-order kinetic constant was 4% larger and the use of electron donor was 16.6% more efficient under NH4+ famine conditions. Sulphidogenesis is robust to transient feeding conditions, but not when applying high loading rates (SBR H2 and H3).

Lovastatin effect in rat neuroblasts of the CNS: inhibition of cap-dependent translation.

Mevalonate biosynthesis pathway is important in cell growth and survival and its blockade by 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, arrest brain neuroblasts growth and induce apoptosis. Translation is among the main biochemical mechanisms that controls gene expression and therefore cell growth or apoptosis. In the CNS, translation regulates synaptic plasticity. Thus, our aim was to investigate the effect of lovastatin in protein translation in rat neuroblasts of the CNS and the biochemical pathways involved. Lovastatin treatment in rat brain neuroblasts causes a significant time- and concentration-inhibition of protein synthesis, which is partially mediated by phosphatydilinositol 3-kinase/mammalian target of rapamycin (mTOR) pathway inhibition. Lovastatin treatment decreases the phosphorylation state of mTOR substrates, p70S6K and eukaryotic translation initiation factor (eIF) 4E-binding protein 1 and simultaneously increases eIF4E-binding protein 1 in a time-dependent manner. Concomitantly, lovastatin causes a decrease in eIF4G cellular amount, which is partially mediated by caspase(s) activity excluding caspase 3. These biochemical pathways affected by lovastatin might explain the protein translation inhibition observed in neuroblasts. Cycloheximide treatment, which blocked protein synthesis, does not induce neuroblasts apoptosis. Therefore, we suggest that lovastatin-induced protein synthesis inhibition might not contribute to the concomitant neuroblasts apoptosis previously observed.

Addition of granular activated carbon and trace elements to favor volatile fatty acid consumption during anaerobic digestion of food waste.

The effect of supplementing granular activated carbon and trace elements on the anaerobic digestion performance of consecutive batch reactors treating food waste was investigated. The results from the first batch suggest that addition of activated carbon favored biomass acclimation, improving acetic acid consumption and enhancing methane production. Adding trace elements allowed a faster consumption of propionic acid. A second batch proved that a synergy existed when activated carbon and trace elements were supplemented simultaneously. The degradation kinetics of propionate oxidation were particularly improved, reducing significantly the batch duration and improving the average methane productivities. Addition of activated carbon favored the growth of archaea and syntrophic bacteria, suggesting that interactions between these microorganisms were enhanced. Interestingly, microbial analyses showed that hydrogenotrophic methanogens were predominant. This study shows for the first time that addition of granular activated carbon and trace elements may be a feasible solution to stabilize food waste anaerobic digestion.

Cooperative growth of Geobacter sulfurreducens and Clostridium pasteurianum with subsequent metabolic shift in glycerol fermentation.

Interspecies electron transfer is a common way to couple metabolic energy balances between different species in mixed culture consortia. Direct interspecies electron transfer (DIET) mechanism has been recently characterised with Geobacter species which couple the electron balance with other species through physical contacts. Using this mechanism could be an efficient and cost-effective way to directly control redox balances in co-culture fermentation. The present study deals with a co-culture of Geobacter sulfurreducens and Clostridium pasteurianum during glycerol fermentation. As a result, it was shown that Geobacter sulfurreducens was able to grow using Clostridium pasteurianum as sole electron acceptor. C. pasteurianum metabolic pattern was significantly altered towards improved 1,3-propanediol and butyrate production (+37% and +38% resp.) at the expense of butanol and ethanol production (-16% and -20% resp.). This metabolic shift was clearly induced by a small electron uptake that represented less than 0.6% of the electrons consumed by C. pasteurianum. A non-linear relationship was found between G. sulfurreducens growth (i.e the electrons transferred between the two species) and the changes in C. pasteurianum metabolite distribution. This study opens up new possibilities for controlling and increasing specificity in mixed culture fermentation.

Conservation of acquired morphology and community structure in aged biofilms after facing environmental stress.

The influence of growth history on biofilm morphology and microbial community structure is poorly studied despite its important role for biofilm development. Here, biofilms were exposed to a change in hydrodynamic conditions at different growth stages and we observed how biofilm age affected the change in morphology and bacterial community structure. Biofilms were developed in two bubble column reactors, one operated under constant shear stress and one under variable shear stress. Biofilms were transferred from one reactor to the other at different stages in their development by withdrawing and inserting the support medium from one reactor to the other. The developments of morphology and microbial community structure were followed by image analysis and molecular tools. When transferred early in biofilm development, biofilms adapted to the new hydrodynamic conditions and adopted features of the biofilm already developed in the receiving reactor. Biofilms transferred at a late state of biofilm development continued their initial trajectories of morphology and community development even in a new environment. These biofilms did not immediately adapt to their new environment and kept features acquired during their early growth phase, a property we called memory effect.

Biofilm development during the start-up period of anaerobic biofilm reactors: the biofilm Archaea community is highly dependent on the support material.

To evaluate the impact of the nature of the support material on its colonization by a methanogenic consortium, four substrata made of different materials: polyvinyl chloride, 2 polyethylene and polypropylene were tested during the start-up of lab-scale fixed-film reactors. The reactor performances were evaluated and compared together with the analysis of the biofilms. Biofilm growth was quantified and the structure of bacterial and archaeal communities were characterized by molecular fingerprinting profiles (capillary electrophoresis-single strand conformation polymorphism). The composition of the inoculum was shown to have a major impact on the bacterial composition of the biofilm, whatever the nature of the support material or the organic loading rate applied to the reactors during the start-up period. In contrast, the biofilm archaeal populations were independent of the inoculum used but highly dependent on the support material. Supports favouring Archaea colonization, the limiting factor in the overall process, should be preferred.

Recurrence after gross-total resection of low-grade pediatric brain tumors: the frequency and timing of postoperative imaging.

OBJECT: Low-grade glial and glioneuronal brain tumors are frequently encountered in the pediatric population and can be effectively treated by resection. The authors aimed to use imaging to evaluate how often tumors recurred and to determine if recurrences were associated with any clinical symptoms, along with the financial costs of imaging, in patients with radiographically proven gross-total resection (GTR) at Boston Children's Hospital. These data were assessed to propose guidelines regarding postoperative surveillance. METHODS: The authors performed a retrospective cohort analysis of the Pediatric Brain Tumor Program database from 1993 to 2003 to identify patients with glial or glioneuronal tumors initially evaluated at Boston Children's Hospital. Among the 888 patients evaluated for any type of brain tumor during this period, 67 patients had WHO Grade I glial or glioneuronal lesions with radiographically proven GTR and available follow-up data. The frequency and timing of postoperative imaging was compared with the institutional protocol. Recurrence-free survival was calculated using the Kaplan-Meier method. Financial costs of imaging were available from 2001 to 2009 and were averaged to extrapolate the postoperative surveillance costs. RESULTS: Among the 67 patients with GTR, 13 recurrences were detected radiographically with a mean time to recurrence of 32.4 months (range 2.9-128.5 months). The mean duration of follow-up after surgery was 6.6 years. The recurrence-free survival at 2 and 5 years after GTR for all low-grade glial and glioneuronal tumors was 0.90 (95% CI 0.82-0.97) and 0.82 (95% CI 0.73-0.92), respectively. No clinical symptoms were associated with any of the recurrences, and no deaths occurred. Under the institutional protocol of surveillance imaging, the estimated cost per recurrence at 5 years was $104,094 per patient. The proposed protocol would reduce the number of MR scans in the first 5 years from 10 to 5, providing a potential cost savings of $52,047 per recurrence. CONCLUSIONS: Given the slow-growing, clinically asymptomatic nature of low-grade glial and glioneuronal tumors coupled with the financial and psychological costs of repeated imaging, the authors propose a postoperative surveillance MRI schedule that is less intensive than current institutional practice.