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.

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.

In geothermischen Anlagen können Biofilme die Mineralbildung und die Injektivität von Bohrungen sowie die Materialbeständigkeit beeinträchtigen. In drei bezüglich Temperatur und Salinität sehr unterschiedlichen Anlagen waren Organismen des Schwefelkreislaufs an Betriebsstörungen beteiligt: Die erhöhte Abundanz von Sulfat-reduzierenden Bakterien (SRB) auf der kalten Seite eines Wärmespeichers wies auf deren Beteiligung an der Korrosion und der Abnahme der Injektivität hin. In allen Anlagen führte der Zutritt von Sauerstoff bzw. der Eintrag von Nitrat zu einer temporären Zunahme Schwefel-oxidierender Bakterien (SOB) und hat vermutlich Korrosionsprozesse beschleunigt. Außerdem hatte in einem Kältespeicher die temporäre Zunahme der SOB ein Filterclogging zur Folge. Aufgrund ihrer entscheidenden Rolle bei mikrobiell induzierter Korrosion (MIC) weisen Änderungen in der Abundanz von SOB und SRB auf die Ursachen mikrobiell bedingter Störungen hin. Zur Beseitigung der Störungen wurden temporäre Erhöhungen der Temperatur, Säuerungen sowie die Zugabe von Wasserstoffperoxid (H2O2) oder Nitrat in den Anlagen getestet und aus mikrobiologischer Sicht bewertet.

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, 14C-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, 14CO2-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 14C 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 14C 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.

This paper describes microbial metabolic processes that are considered to be relevant for the technical reliability of a geothermal heat store. The study reports on changes of the microbial community composition in geothermal well fluids of different temperatures and after plant downtimes monitored by genetic fingerprinting. Stagnant conditions favored the enrichment of bacteria, sulfate reducers (SRB), and sulfur oxidizers (SOB) in the well. Furthermore higher concentrations of DOC, SO42-, H2S, and H2 were detected in the first fluids produced after plant downtime. The increased abundance of SOB indicated oxygen ingress during plant downtime. The interaction of SRB and SOB might have further enhanced corrosion and scaling processes. A mobile bypass system installed at the site will help to understand the processes occurring in the well and to study biofilm formation and corrosion rates at different temperatures.

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.

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 14C-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-14C-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 14C-labeled glycerol L−1 (1 % v/v) resulted in the production of 17.1 mg 14C-Cry1Ab L−1 cultivation medium. 14C 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 14C originated from Cry1Ab. In the presence of 2.04 MBq 14C-labeled carbon sources, a specific activity of up to 268 Bq mg−1 14C-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 14C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple 14C-labeled carbon source. It also provides a general strategy to produce 14C-labeled proteins useful for soil metabolic studies.

In this study, the operation of a cold store, located in 30–60 m depth in the North German Basin, was investigated by direct counting of bacteria and genetic fingerprinting analysis. Quantification of microbes accounted for 1 to 10⋅105 cells per ml fluid with minor differences in the microbial community composition between well and process fluids. The detected microorganisms belong to versatile phyla Proteobacteria and Flavobacteria. In addition to routine plant operation, a phase of plant malfunction caused by filter clogging was monitored. Increased abundance of sulfur-oxidizing bacteria indicated a change in the supply of electron acceptors, however, no changes in the availability of electron acceptors like nitrate or oxygen were detected. Sulfur- and iron-oxidizing bacteria played essential roles for the filter lifetimes at the topside facility and the injectivity of the wells due to the formation of biofilms and induced mineral precipitations. In particular, sulfur-oxidizing Thiothrix generated filamentous biofilms were involved in the filter clogging.

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.

A three-year experimental Held study with a genetically engineered Bt maize (event MON88017) and ;three conventionally bred cultivars was conducted to quantify the recombinant Cry3Bb1 protein released into soil and detect effects on the diversity of soil bacteria. Protein extraction and an enzyme-linked immunosorbent assay (ELISA) allowed a threshold detection of 0.01 ng Cry3Bb1 g(-1) soil. The Maximum amount found in field plots with Bt maize was 1.0 ng Cry3Bb1 g(-1) rhizosphere soil. Average concentrations during the growing seasons varied between years from 0.07 to 0.29 ng g(-1). No accumulation of Cry3Bb1 in soil occurred over the three growing seasons. Four weeks after harvest, the major Cry3Bb1 reservoirs on the field were the remaining root stubbles, but their Cry3Bb1 concentration declined by 98.30-99.99% in the following seven months. During the three consecutive years of study there were never significant differences between the rhizosphere bacterial community structure of the Bt maize and the other cultivars, as detected by cultivation independent profiling of PCR-amplified 16S rRNA genes. The low concentrations of soil extractable Cry3Bb1, its degradation in decaying roots, and the lack of effects on rhizosphere bacteria give no indications of adverse effects of MON88017 cultivation on soil ecology. (C) 2009 Elsevier Masson SAS. All rights reserved.

A field study was conducted with genetically modified sinorhizobium meliloti strains L1 (RecA-) and L33 (RecA+), both tagged with the firefly luciferase luc gene as an identification marker. The strains' fate was studied over a time period of five years. Both strains were rapidly outcompeted for alfalfa nodulation by an indigenous population. In summary, this study demonstrates the usefulness of tagging bacteria designed for environmental releases by the firefly luciferase gene and the high resilience of soil bacteria to allow the establishment of foreign bacterial populations. This work was supported by grants of the German Federal Ministry of Education and Research (BMBF). We thank Penny Hirsch for valuable discussion concerning the preparation of the peat-based inoculants. The contribution of the Bayer-ische Staatsministerium fur Ernahrung, Landwirtschaft und Forsten for making available the field release site in Stras-smoos is gratefully acknowledged. We thank Rudolf Beck and the team of the Strassmoos experimental plant station for their excellent support during the field study.

Despite the fact that the number of deliberate field releases of marker gene-tagged bacteria is still relatively low compared to field studies with genetically engineered plants, we can already look back over a significant amount of work which was conducted during the last 20 years in a number of different countries and continents. Most of these studies were conducted just to follow the fate of inoculated cells into the environment and to test hypotheses about their behaviour as predicted by fine-scale molecular analyses and soil microcosm studies. Genetic engineering has been the crucial technique to equip bacterial inoculants with a selective and/or detection marker and thereby allowed the monitoring of their fate in the environment. Genetic engineering, however, is also the most powerful technology for improving inoculants, as it enables the development of much broader and more targeted solutions than classical mutation techniques. The discussion about the potential risks connected with the deliberate release of such optimised inoculants is as important as it was 20 years ago. The experiences made with the field releases thus far are one of the most important sources of information for discussion and risk assessment on a case-by-case basis. The most important message from these studies is that no such release has caused any known damage to human health or to ecosystem functions. On the one hand, this is not an encouragement to extrapolate a zero-risk expectation for the future. On the other hand, it is a clear indication of how valuable such small-scale field releases were and can be, to extend our knowledge about the interactions between microorganisms and their environment and to develop products that contribute to a sustainable use of biological resources in agriculture. It remains a challenge for environmental microbiologists not only to unravel and optimise the highly beneficial properties of microorganisms for environmental applications, but also to share this fascinating and important endeavour with the public.