Abstract. This study explores the applicability of DNA analyses for the characterization of primary biogenic aerosol (PBA) particles in the atmosphere. Samples of fine particulate matter (PM 2.5 ) and total suspended particulates (TSP) have been collected on different types of filter materials at urban, rural, and high-alpine locations along an altitude transect in the south of Germany (Munich, Hohenpeissenberg, Mt. Zugspitze).From filter segments loaded with about one milligram of air particulate matter, DNA could be extracted and DNA sequences could be determined for bacteria, fungi, plants and animals. Sequence analyses were used to determine the identity of biological organisms, and terminal restriction fragment length polymorphism analyses (T-RFLP) were applied to estimate diversities and relative abundances of bacteria. Investigations of blank and background samples showed that filter materials have to be decontaminated prior to use, and that the sampling and handling procedures have to be carefully controlled to avoid artifacts in the analyses.Mass fractions of DNA in PM 2.5 were found to be around 0.05% in urban, rural, and high-alpine aerosols. The average concentration of DNA determined for urban air was on the order of ∼7 ng m −3 , indicating that human adults may inhale about one microgram of DNA per day (corresponding to ∼10 8 haploid bacterial genomes or ∼10 5 haploid human genomes, respectively).Most of the bacterial sequences found in PM 2.5 were from Proteobacteria (42) and some from Actinobacteria (10) and Firmicutes (1). The fungal sequences were characteristic for Ascomycota (3) and Basidiomycota (1), which are known to actively discharge spores into the atmosphere. The plant Correspondence to: V. R. Després (despres@mpch-mainz.mpg.de) sequences could be attributed to green plants (2) and moss spores (2), while animal DNA was found only for one unicellular eukaryote (protist).Over 80% of the 53 bacterial sequences could be matched to one of the 19 T-RF peaks found in the PM 2.5 samples, but only 40% of the T-RF peaks did correspond to one of the detected bacterial sequences. The results demonstrate that the T-RFLP analysis covered more of the bacterial diversity than the sequence analysis. Shannon-Weaver indices calculated from both sequence and T-RFLP data indicate that the bacterial diversity in the rural samples was higher than in the urban and alpine samples. Two of the bacterial sequences (Gammaproteobacteria) and five of the T-RF peaks were found at all sampling locations.

No abstract

The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture.

Methane is an important end product of degradation of organic matter in anoxic lake sediments. Methane is mainly produced by either reduction of CO₂ or cleavage of acetate involving different methanogenic archaea. The contribution of the different methanogenic paths and of the diverse bacteria and archaea involved in CH₄ production exhibits a large variability that is not well understood. Lakes in tropical areas, e.g. in Brazil, are wetlands with high potential impact on the global CH₄ budget. However, they have hardly been studied with respect to methanogenesis. Therefore, we used samples from 16 different lake sediments in the Pantanal and Amazon region of Brazil to measure production of CH₄, CO₂, analyze the content of ¹³C in the products and in intermediately formed acetate, determine the abundance of bacterial and archaeal microorgansisms and their community composition and diversity by targeting the genes of bacterial and archaeal ribosomal RNA and of methyl coenzyme M reductase, the key enzyme of methanogenic archaea. These experiments were done in the presence and absence of methyl fluoride, an inhibitor of acetoclastic methanogenesis. While production rates of CH₄ and CO₂ were correlated to the content of organic matter and the abundance of archaea in the sediment, values of ¹³C in acetate, CO₂, and CH₄ were related to the ¹³C content of organic matter and to the path of CH₄ production with its intrinsic carbon isotope fractionation. Isotope fractionation was small (average 10‰) for conversion of C_org to acetate-methyl, which was hardly further fractionated during CH₄ production. However, fractionation was strong for CO₂ conversion to CH₄ (average 75‰), which generally accounted for >50% of total CH₄ production. Canonical correspondence analysis did not reveal an effect of microbial community composition, despite the fact that it exhibited a pronounced variability among the different sediments

The community structure of methanogenic Archaea on anoxically incubated rice roots was investigated by amplification, sequencing, and phylogenetic analysis of 16S rRNA and methyl-coenzyme M reductase (mcrA) genes. Both genes demonstrated the presence of Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae, Methanosaetaceae, and Rice cluster I, an uncultured methanogenic lineage. The pathway of CH4 formation was determined from the 13C-isotopic signatures of the produced CH4, CO2 and acetate. Conditions and duration of incubation clearly affected the methanogenic community structure and the pathway of CH4 formation. Methane was initially produced from reduction of CO2 exclusively, resulting in accumulation of millimolar concentrations of acetate. Simultaneously, the relative abundance of the acetoclastic methanogens (Methanosarcinaceae, Methanosaetaceae), as determined by T-RFLP analysis of 16S rRNA genes, was low during the initial phase of CH4 production. Later on, however, acetate was converted to CH4 so that about 40% of the produced CH4 originated from acetate. Most striking was the observed relative increase of a population of Methanosarcina spp. (but not of Methanosaeta spp.) briefly before acetate concentrations started to decrease. Both acetoclastic methanogenesis and Methanosarcina populations were suppressed by high phosphate concentrations, as observed under application of different buffer systems. Our results demonstrate the parallel change of microbial community structure and function in a complex environment, i.e., the increase of acetoclastic Methanosarcina spp. when high acetate concentrations become available.

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4), acetate, propionate and butyrate when incubated under anoxic conditions. Acetate production was most pronounced with a maximum rate (mean+/-standard error; four different root preparations) of 3.4+/-0.6 µmol h(-1) g-dry weight(-1) roots, compared to 0.45+/-0.13, 0.06+/-0.03, and 0.04+/-0.01 µmol h(-1) g-dw(-1) for propionate, butyrate and CH(4)1 kPa after one day of incubation. Then it decreased and reached more or less constant concentrations of about 50-80 Pa after about 7-8 days. Hydrogen partial pressures were always high enough to allow exergonic methanogenesis (DeltaG=-67 to -98 kJ mol(-1) CH(4)) and exergonic homoacetogenesis (DeltaG=-18 to -48 kJ mol(-1) acetate) from H(2) plus CO(2). Radioactive bicarbonate/CO(2) was incorporated into CH(4), acetate and propionate. The specific radioactivities of the products indicated that CH(4) was exclusively produced from H(2)/CO(2) confirming a previous study. The contribution of CO(2) to the production of acetate and propionate was 32-39% and 42-61%, respectively, assuming that each carbon atom was equally labeled. Propionate also became radioactively labeled, when the roots were incubated with either [1-(14)C]acetate or [2-(14)C]acetate accounting for 60-76% of total propionate production. Reductive formation of propionate was thermodynamically favorable both from H(2) plus acetate plus CO(2) (DeltaG=-15 to -38 kJ mol(-1) propionate) and from H(2) plus CO(2) (DeltaG=-34 to -85 kJ mol(-1) propionate). A substantial fraction of propionate was apparently reductively formed from acetate and/or CO(2). In conclusion, our results demonstrate an intensive anaerobic dark metabolism of CO(2) on washed rice roots with reduction of CO(2) contributing significantly to the production of acetate, propionate and CH(4). The CO(2) reduction seemed to be driven by decay and fermentation of root material.

No abstract

The microbial community in anoxic rice field soil produces CH(4) over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH(4) production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC-1) changes to almost complete dominance of RC-1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH(4) production measuring δ(13)C in CH(4) and CO(2) and calculating the apparent fractionation factor (α(app)) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T-RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T-RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre-incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well-defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42-46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.