Process Recovery after CaO Addition Due to Granule Formation in a CSTR Co-Digester-A Tool to Influence the Composition of the Microbial Community and Stabilize the Process?

The composition, structure and function of granules formed during process recovery with calcium oxide in a laboratory-scale fermenter fed with sewage sludge and rapeseed oil were studied. In the course of over-acidification and successful process recovery, only minor changes were observed in the bacterial community of the digestate, while granules appeared during recovery. Fluorescence microscopic analysis of the granules showed a close spatial relationship between calcium and oil and/or long chain fatty acids. This finding further substantiated the hypothesis that calcium precipitated with carbon of organic origin and reduced the negative effects of overloading with oil. Furthermore, the enrichment of phosphate minerals in the granules was shown, and molecular biological analyses detected polyphosphate-accumulating organisms as well as methanogenic archaea in the core. Organisms related to Methanoculleus receptaculi were detected in the inner zones of a granule, whereas they were present in the digestate only after process recovery. This finding indicated more favorable microhabitats inside the granules that supported process recovery. Thus, the granule formation triggered by calcium oxide addition served as a tool to influence the composition of the microbial community and to stabilize the process after overloading with oil.

Effects of plant downtime on the microbial community composition in the highly saline brine of a geothermal plant in the North German Basin.

The microbial biocenosis in highly saline fluids produced from the cold well of a deep geothermal heat store located in the North German Basin was characterized during regular plant operation and immediately after plant downtime phases. Genetic fingerprinting revealed the dominance of sulfate-reducing bacteria (SRB) and fermentative Halanaerobiaceae during regular plant operation, whereas after shutdown phases, sequences of sulfur-oxidizing bacteria (SOB) were also detected. The detection of SOB indicated oxygen ingress into the well during the downtime phase. High 16S ribosomal RNA (rRNA) and dsrA gene copy numbers at the beginning of the restart process showed an enrichment of bacteria, SRB, and SOB during stagnant conditions consistent with higher concentrations of dissolved organic carbon (DOC), sulfate, and hydrogen sulfide in the produced fluids. The interaction of SRB and SOB during plant downtimes might have enhanced the corrosion processes occurring in the well. It was shown that scale content of fluids was significantly increased after stagnant phases. Moreover, the sulfur isotopic signature of the mineral scales indicated microbial influence on scale formation.

Fate of the insecticidal Cry1Ab protein of GM crops in two agricultural soils as revealed by ¹4C-tracer studies.

Insecticidal delta-endotoxins of Bacillus thuringiensis are among the most abundant recombinant proteins released by genetically modified (GM) crops into agricultural soils worldwide. However, there is still controversy about their degradation and accumulation in soils. In this study, (14)C-labelled Cry1Ab protein was applied to soil microcosms at two concentrations (14 and 50 µg g(-1) soil) to quantify the mineralization of Cry1Ab, its incorporation into the soil microbial biomass, and its persistence in two soils which strongly differed in their texture but not in silt or pH. Furthermore, ELISA was used to quantify Cry1Ab and its potential immunoreactive breakdown products in aqueous soil extracts. In both soils, (14)CO2-production was initially very high and then declined during a total monitoring period of up to 135 days. A total of 16 to 23 % of the (14)C activity was incorporated after 29 to 37 days into the soil microbial biomass, indicating that Cry1Ab protein was utilized by microorganisms as a growth substrate. Adsorption in the clay-rich soil was the most important factor limiting microbial degradation; as indicated by higher degradation rates in the more sandy soil, extremely low concentrations of immunoreactive Cry1Ab molecules in the soils' aqueous extracts and a higher amount of (14)C activity bound to the soil with more clay. Ecological risk assessments of Bt-crops should therefore consider that the very low concentrations of extractable Cry1Ab do not reflect the actual elimination of the protein from soils but that, on the other hand, desorbed proteins mineralize quickly due to efficient microbial degradation.

Exposure to silver nanoparticles induces size- and dose-dependent oxidative stress and cytotoxicity in human colon carcinoma cells.

The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most frequently utilized nanomaterials in consumer products; therefore, a comprehensive understanding of their toxicity is necessary. In particular, information about the cellular uptake and size dependence of AgNPs is insufficient. In this study, we evaluated the size-dependent effects of AgNPs by treating the human LoVo cell line, an intestinal epithelium model, with spherical AgNPs of well-defined sizes (10, 20, 40, 60 and 100nm). The cellular uptake was visualized by confocal laser scanning microscopy, and various cytotoxicity parameters were analyzed in a size- and dose-dependent manner. In addition, the cellular proteomic response to 20 and 100nm AgNPs was investigated to increase the understanding of potential mechanisms of action. Our data indicated that cellular uptake and toxicity were regulated by size; smaller particles easily penetrated the cells, and 100nm particles did not. It was hypothesized that this size-dependent effect resulted from the stimulation of a signaling cascade that generated ROS and inflammatory markers, leading to mitochondrial dysfunction and subsequently inducing apoptosis. By contrast, the cell proliferation, was independent of AgNPs particle size, indicating a differentially regulated, ROS-independent pathway.

Insights into the cellular response triggered by silver nanoparticles using quantitative proteomics.

The use of nanoparticles in foods, materials, and clinical treatments has increased dramatically in the past decade. Because of the possibility of human exposure to nanoparticles, there is an urgent need to investigate the molecular mechanisms underlying the cellular responses that might be triggered. Such information is necessary to assess potential health risks arising from the use of nanoparticles, and for developing new formulations of next generation nanoparticles for clinical treatments. Using mass spectrometry-based proteomic technologies and complementary techniques (e.g., Western blotting and confocal laser scanning microscopy), we present insights into the silver nanoparticle-protein interaction in the human LoVo cell line. Our data indicate that some unique cellular processes are driven by the size. The 100 nm nanoparticles exerted indirect effects via serine/threonine protein kinase (PAK), mitogen-activated protein kinase (MAPK), and phosphatase 2A pathways, and the 20 nm nanoparticles induced direct effects on cellular stress, including generation of reactive oxygen species and protein carbonylation. In addition, we report that proteins involved in SUMOylation were up-regulated after exposure to 20 nm silver nanoparticles. These results were further substantiated by the observation of silver nanoparticles entering the cells; however, data indicate that this was determined by the size of the nanoparticles, since 20 nm particles entered the cells while 100 nm particles did not.

Thermal effects on microbial composition and microbiologically induced corrosion and mineral precipitation affecting operation of a geothermal plant in a deep saline aquifer.

The microbial diversity of a deep saline aquifer used for geothermal heat storage in the North German Basin was investigated. Genetic fingerprinting analyses revealed distinct microbial communities in fluids produced from the cold and warm side of the aquifer. Direct cell counting and quantification of 16S rRNA genes and dissimilatory sulfite reductase (dsrA) genes by real-time PCR proved different population sizes in fluids, showing higher abundance of bacteria and sulfate reducing bacteria (SRB) in cold fluids compared with warm fluids. The operation-dependent temperature increase at the warm well probably enhanced organic matter availability, favoring the growth of fermentative bacteria and SRB in the topside facility after the reduction of fluid temperature. In the cold well, SRB predominated and probably accounted for corrosion damage to the submersible well pump and iron sulfide precipitates in the near wellbore area and topside facility filters. This corresponded to lower sulfate content in fluids produced from the cold well as well as higher content of hydrogen gas that was probably released from corrosion, and maybe favored growth of hydrogenotrophic SRB. This study reflects the high influence of microbial populations for geothermal plant operation, because microbiologically induced precipitative and corrosive processes adversely affect plant reliability.

Production of the 14C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations.

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce (14)C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-(14)C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4 mg C L(-1). Concentrations of 12.6 g (14)C-labeled glycerol L(-1) (1 % v/v) resulted in the production of 17.1 mg (14)C-Cry1Ab L(-1) cultivation medium. (14)C mass balances showed that approx. 50 % of the label was lost by respiration and 20 % remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05 % of the total (14)C originated from Cry1Ab. In the presence of 2.04 MBq (14)C-labeled carbon sources, a specific activity of up to 268 Bq mg(-1) (14)C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that (14)C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple (14)C-labeled carbon source. It also provides a general strategy to produce (14)C-labeled proteins useful for soil metabolic studies.

Spatial heterogeneity of dechlorinating bacteria and limiting factors for in situ trichloroethene dechlorination revealed by analyses of sediment cores from a polluted field site.

Microbiological analyses of sediment samples were conducted to explore potentials and limitations for bioremediation of field sites polluted with chlorinated ethenes. Intact sediment cores, collected by direct push probing from a 35-ha contaminated area, were analyzed in horizontal layers. Cultivation-independent PCR revealed Dehalococcoides to be the most abundant 16S rRNA gene phylotype with a suspected potential for reductive dechlorination of the major contaminant trichloroethene (TCE). In declining abundances, Desulfitobacterium, Desulfuromonas and Dehalobacter were also detected. In TCE-amended sediment slurry incubations, 66% of 121 sediment samples were dechlorinating, among them one-third completely and the rest incompletely (end product cis-1,2-dichloroethene; cDCE). Both PCR and slurry analyses revealed highly heterogeneous horizontal and vertical distributions of the dechlorination potentials in the sediments. Complete reductive TCE dechlorination correlated with the presence of Dehalococcoides, accompanied by Acetobacterium and a relative of Trichococcus pasteurii. Sediment incubations under close to in situ conditions showed that a low TCE dechlorination activity could be stimulated by 7 mg L(-1) dissolved carbon for cDCE formation and by an additional 36 mg carbon (lactate) L(-1) for further dechlorination. The study demonstrates that the highly heterogeneous distribution of TCE degraders and their specific requirements for carbon and electrons are key issues for TCE degradation in contaminated sites.