Microwave and ultrasound pre-treatments influence microbial community structure and digester performance in anaerobic digestion of waste activated sludge.

Comparative analyses of bacterial and archaeal community structures and dynamics in three biogas digesters during start-up and subsequent operation using microwaved, ultrasonicated or untreated waste activated sludge were performed based on 454 pyrosequencing datasets of part of 16S ribosomal RNA sequences and quantitative PCR. The pre-treatment increased the solubility, and thus the availability of the substrate for microbial degradation and significantly affected the succession of the anaerobic community structure over the course of the digestion. Bacteroidetes, Proteobacteria and Firmicutes were the dominant phyla in all digesters throughout operation. Proteobacteria decreased in relative abundance from 23-26 % to 11-13 % in association with enhanced substrate availability. Negative correlations between relative abundance of Alpha-, Beta- and Gammaproteobacteria and the substrate availability and/or biogas production were disclosed in statistical analyses. Clostridiales was the dominant order in Firmicutes, and Clostridiales, Clostridia and Firmicutes relative abundance and richness were shown to positively correlate with substrate availability and biogas generation. Methanogenic communities had a fairly restricted structure, highly dominated by Methanosaeta and Methanobrevibacter phylotypes. A gradual decline in Methanobrevibacter and increased representation of Methanosaeta concilii over time were particularly apparent in the digester receiving untreated waste activated sludge, whereas more diversified archaeal communities were maintained in the pre-treatment digesters. The quantitative PCR analyses revealed a methanogenic community distribution that coincided with the 454 pyrosequencing data.

Extremophiles survival to simulated space conditions: an astrobiology model study.

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

High-performance genetically targetable optical neural silencing by light-driven proton pumps.

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of 'optogenetic' voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations.

Light-induced modulation of protein dynamics during the photocycle of bacteriorhodopsin.

Knowledge about the dynamical properties of a protein is of essential importance for understanding the structure-dynamics-function relationship at the atomic level. So far, however, the correlation between internal protein dynamics and functionality has only been studied indirectly in steady-state experiments by variation of external parameters like temperature and hydration. In the present study we describe a novel type of (laser-neutron) pump-probe experiment, which combines in situ optical activation of the biological function of a membrane protein with a time-dependent monitoring of the protein dynamics using quasielastic neutron scattering. As a first successful application we present data obtained selectively in the ground state and in the M-intermediate of bacteriorhodopsin (BR). Temporary alterations in both localized reorientational protein motions and harmonic vibrational dynamics have been observed during the photocycle of BR. This observation is a direct proof for the functional significance of protein structural flexibility, which is correlated with the large-scale structural changes in the protein structure occurring during the M-intermediate. We anticipate that functionally important modulations of protein dynamics as observed here are of relevance for most other proteins exhibiting conformational transitions in the time course of functional operation.

Light sensitivity of methanogenic archaebacteria.

Representatives of four families of methanogenic archaebacteria (archaea), Methanobacterium thermoautotrophicum delta H, Methanobacterium thermoautotrophicum Marburg, Methanosarcina acetivorans, Methanococcus voltae, and Methanomicrobium mobile, were found to be light sensitive. The facultative anaerobic eubacteria Escherichia coli and Salmonella typhimurium, however, were tolerant of light when grown anaerobically under identical light conditions. Interference filters were used to show that growth of the methanogens is inhibited by light in the blue end of the visible spectrum (370 to 430 nm).

Molecular cloning and characterization of the recA gene of Methylomonas clara and construction of recA deficient mutant.

The recA gene of the methylotrophic bacterium Methylomonas clara has been isolated from a genomic library by hybridization with the Escherichia coli recA gene. Its complete nucleotide sequence consists of 1029 bp encoding a polypeptide of 342 amino acids. Nucleotide sequence analysis of the M. clara recA gene revealed extensive homologies to recA genes from E. coli and Pseudomonas aeruginosa. Part of the physiological activity of the M. clara RecA protein has become evident in that E. coli recA mutant HB101 is complemented. The cloned recA gene has been modified in vitro by site-specific mutagenesis and by insertion of a kanamycin-resistance gene cassette into the recA coding sequence. M. clara recA mutants were obtained by replacement of the active recA gene by an in-vitro inactivated gene copy.

Influence of corrinoid antagonists on methanogen metabolism.

Iodopropane inhibited cell growth and methane production when Methanobacterium thermoautotrophicum, Methanobacterium formicicum, and Methanosarcina barkeri were cultured on H2-CO2. Iodopropane (40 microM) inhibited methanogenesis (30%) and growth (80%) when M. barkeri was cultured mixotrophically on H2-CO2-methanol. The addition of acetate to the medium prevented the observed iodopropane-dependent inhibition of growth. The concentrations of iodopropane that caused 50% inhibition of growth of M. barkeri on either H2-CO2, H2-CO2-methanol, methanol, and acetate were 112 +/- 6, 24 +/- 2, 63 +/- 11, and 4 +/- 1 microM, respectively. Acetate prevented the iodopropane-dependent inhibition of one-carbon metabolism. Cultivation of M. barkeri on H2-CO2-methanol in bright light also inhibited growth and methanogenesis to a greater extent in the absence than in the presence of acetate in the medium. Acetate was the only organic compound examined that prevented iodopropane-dependent inhibition of one-carbon metabolism in M. barkeri. The effect of iodopropane and acetate on the metabolic fates of methanol and carbon dioxide was determined with 14C tracers when M. barkeri was grown mixotrophically on H2-CO2-methanol. The addition of iodopropane decreased the contribution of methanol to methane and cell carbon while increasing the contribution of CO2 to cell carbon. Regardless of iodopropane, acetate addition decreased the contribution of methanol and CO2 to cell carbon without decreasing their contribution to methane. The corrinoid antagonists, light and iodopropane, appeared most specific for methanogen metabolic reactions involved in acetate synthesis from one-carbon compounds and acetate catabolism.