Francisco Vazquez

Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra-and trichloroethene in an anaerobic respiration

Abstract The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H2 as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 ± 0.3 mol%. The cell wall consisted of type-A3γ peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus.

Characterization of the Corrinoid Iron-Sulfur Protein Tetrachloroethene Reductive Dehalogenase of Dehalobacter restrictus

ABSTRACT The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ± 1 kDa, whereas the native molecular mass was 71± 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-β-d-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0± 0.1 mol of cobalamin, 0.6 ± 0.02 mol of cobalt, 7.1± 0.6 mol of iron, and 5.8 ± 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ± 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The Km values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4± 3.2, 23.7 ± 5.2, and 47 ± 10 μM, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria.

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N2O. More than 50% of the 14C from ring-labelled toluene was released as CO2, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N2O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [14C]toluene was transformed to [14C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.

Evidence for anaerobic oxidation of methane in sediments of a freshwater system (Lago di Cadagno).

Anaerobic oxidation of methane (AOM) has been investigated in sediments of a high alpine sulfate-rich lake. Hot spots of AOM could be identified based on geochemical and isotopic evidence. Very high fractionation of methane (α=1.031) during oxidation was observed in the uppermost sediment layers, where methane is oxidized most likely with sulfate-containing bottom waters. However, we could not exclude that other electron acceptors such as iron, or manganese might also be involved. Light carbon isotope values (δ¹³C = -10‰ vs. Vienna Pee Dee Belemnite [VPDB]) of sedimentary carbonates at 16-20 cm sediment depth are indicative of a zone where methane was oxidized and the resulting bicarbonate ions were used for carbonate precipitation. 16S rRNA gene analysis revealed the presence of sequences belonging to the marine benthic groups B, C, and D and to the recently described clade of AOM-associated archaea (AAA). Catalyzed reporter deposition-FISH analysis revealed a high abundance of Deltaproteobacteria, especially of free-living sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus branch of Deltaproteobacteria in the AOM zone. Here, loose aggregations of AAA cells were found, suggesting that AAA might be responsible for oxidation of methane in Lake Cadagno sediments.

Methane sources and sinks in Lake Kivu

Unique worldwide, Lake Kivu stores enormous amounts of CH 4 and CO 2 . A recent study reported that CH 4 concentrations in the lake have increased by up to 15% in the last 30 years and that accumulation at this rate could lead to catastrophic outgassing by ∼2100. This study investigates the present-day CH 4 formation and oxidation in Lake Kivu. Analyses of 14C and 13C in CH 4 and potential carbon sources revealed that below 260 m, an unusually high ∼65% of the CH 4 originates either from reduction of geogenic CO 2 with mostly geogenic H 2 or from direct inflows of geogenic CH 4 . Aerobic CH 4 oxidation, performed by close relatives of type X CH 4 -oxidizing bacteria, is the main process preventing CH 4 from escaping to the atmosphere. Anaerobic CH 4 oxidation, carried out by CH 4 -oxidizing archaea in the SO 4 2--reducing zone, was also detected but is limited by the availability of sulfate. Changes in 14C CH4 and 13C CH4 since the 1970s suggest that the amount of CH 4 produced from degrading organic material has increased due to higher accumulation of organic matter. This, as well as the sudden onset of carbonates in the 1960s, has previously been explained by three environmental changes: (1) introduction of nonnative fish, (2) amplified subaquatic inflows following hydrological changes, and (3) increased external inputs due to the fast growing population. The resulting enhancement of primary production and organic matter sedimentation likely caused CH 4 to increase. However, given the large proportion of old CH 4 carbon, we cannot exclude an increased inflow of geogenic H 2 or CH 4 . Copyright 2011 by the American Geophysical Union.

Regime Shift and Microbial Dynamics in a Sequencing Batch Reactor for Nitrification and Anammox Treatment of Urine

ABSTRACT The microbial population and physicochemical process parameters of a sequencing batch reactor for nitrogen removal from urine were monitored over a 1.5-year period. Microbial community fingerprinting (automated ribosomal intergenic spacer analysis), 16S rRNA gene sequencing, and quantitative PCR on nitrogen cycle functional groups were used to characterize the microbial population. The reactor combined nitrification (ammonium oxidation)/anammox with organoheterotrophic denitrification. The nitrogen elimination rate initially increased by 400%, followed by an extended period of performance degradation. This phase was characterized by accumulation of nitrite and nitrous oxide, reduced anammox activity, and a different but stable microbial community. Outwashing of anammox bacteria or their inhibition by oxygen or nitrite was insufficient to explain reactor behavior. Multiple lines of evidence, e.g., regime-shift analysis of chemical and physical parameters and cluster and ordination analysis of the microbial community, indicated that the system had experienced a rapid transition to a new stable state that led to the observed inferior process rates. The events in the reactor can thus be interpreted to be an ecological regime shift. Constrained ordination indicated that the pH set point controlling cycle duration, temperature, airflow rate, and the release of nitric and nitrous oxides controlled the primarily heterotrophic microbial community. We show that by combining chemical and physical measurements, microbial community analysis and ecological theory allowed extraction of useful information about the causes and dynamics of the observed process instability.

Evidence for the existence of psychrophilic methanogenic communities in anoxic sediments of deep lakes

ABSTRACT In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5°C), slurries were first incubated at temperatures between 4 and 60°C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6°C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15°C, with maximal rates in slurries kept at 6°C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6°C after preincubation at 30°C.

Bacterial Chitin Hydrolysis in Two Lakes with Contrasting Trophic Statuses

ABSTRACT Chitin, which is a biopolymer of the amino sugar glucosamine (GlcN), is highly abundant in aquatic ecosystems, and its degradation is assigned a key role in the recycling of carbon and nitrogen. In order to study the significance of chitin decomposition in two temperate freshwater lakes with contrasting trophic and redox conditions, we measured the turnover rate of the chitin analog methylumbelliferyl-N,N′-diacetylchitobioside (MUF-DC) and the presence of chitinase (chiA) genes in zooplankton, water, and sediment samples. In contrast to the eutrophic and partially anoxic lake, chiA gene fragments were detectable throughout the oligotrophic water column and chiA copy numbers per ml of water were up to 15 times higher than in the eutrophic waters. For both lakes, the highest chiA abundance was found in the euphotic zone—the main habitat of zooplankton, but also the site of production of easily degradable algal chitin. The bulk of chitinase activity was measured in zooplankton samples and the sediments, where recalcitrant chitin is deposited. Both, chiA abundance and chitinase activity correlated well with organic carbon, nitrogen, and concentrations of particulate GlcN. Our findings show that chitin, although its overall contribution to the total organic carbon is small (∼0.01 to 0.1%), constitutes an important microbial growth substrate in these temperate freshwater lakes, particularly where other easily degradable carbon sources are scarce.

RECENT STUDIES ON SOURCES AND SINKS OF METHANE IN THE BLACK SEA

This study focuses on the influence of gas seepage on methane sources and sinks, aerobic and anaerobic oxidation of methane and the mediating microbial organisms in the Black Sea. We present data from two cruises that took place in 2001 and 2003. Seven stations (two from the shelf, four from the upper and lower slope, and one from the central basin) were compared with respect to methane concentration and isotope signature. The stations differed in methane concentration depending on the location on the slope. A strong change in the concentration and isotopic composition of methane was observed below the oxic/anoxic interface, coinciding with increased levels of archaeal biomarkers (archaeol and sn-2-hydroxy-archaeol). Concentration and isotopic composition of methane in the water column and sediments indicate that sediments from the shelf, slope, and deep basin are only minor sources of methane. The main methane sources are seeps located on the shelf and upper slope, but also in the deep basin. The comparison of two shelf stations with and without methane seepage showed a difference in methane concentrations, isotopic composition and oxidation rates, but the presence of similar methanotrophic microbial assemblages. Also two deep stations at a seep and outside of a seep area were compared, but here methane concentrations and oxidation rates were not different from each other. Anaerobic methane oxidizers (ANME-1 and ANME-2 group) were observed at both stations with slightly higher cell counts at the seep station.