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Microbes living in plant tissues—endophytes—are mainly studied in crop plants where they typically colonize the root apoplast. Trees—a large carbon source with a high capacity for photosynthesis—provide a variety of niches for endophytic colonization. We have earlier identified a new type of plant–endophyte interaction in buds of adult Scots pine, where Methylorubrum species live inside the meristematic cells. The endosymbiont Methylorubrum extorquens DSM13060 significantly increases needle and root growth of pine seedlings without producing plant hormones, but by aggregating around host nuclei. Here, we studied gene expression and metabolites of the pine host induced by M. extorquens DSM13060 infection. Malic acid was produced by pine to potentially boost M. extorquens colonization and interaction. Based on gene expression, the endosymbiont activated the auxin- and ethylene (ET)-associated hormonal pathways through induction of CUL1 and HYL1, and suppressed salicylic and abscisic acid signaling of pine. Infection by the endosymbiont had an effect on pine meristem and leaf development through activation of GLP1-7 and ALE2, and suppressed flowering, root hair and lateral root formation by downregulation of AGL8, plantacyanin, GASA7, COW1 and RALFL34. Despite of systemic infection of pine seedlings by the endosymbiont, the pine genes CUL1, ETR2, ERF3, HYL, GLP1-7 and CYP71 were highly expressed in the shoot apical meristem, rarely in needles and not in stem or root tissues. Low expression of MERI5, CLH2, EULS3 and high quantities of ononitol suggest that endosymbiont promotes viability and protects pine seedlings against abiotic stress. Our results indicate that the endosymbiont positively affects host development and stress tolerance through mechanisms previously unknown for endophytic bacteria, manipulation of plant hormone signaling pathways, downregulation of senescence and cell death-associated genes and induction of ononitol biosynthesis.

Plant growth and productivity depend on the interactions of the plant with the associated rhizosphere microbes. Rhizosphere protists play a significant role in this respect: considerable efforts have been made in the past to reveal the impact of protist-bacteria interactions on the remobilization of essential nutrients for plant uptake, or the grazing induced changes on plant-growth promoting bacteria and the root-architecture. However, the metabolic responses of plants to the presence of protists or to protist-bacteria interactions in the rhizosphere have not yet been analyzed. Here we studied in controlled laboratory experiments the impact of bacterivorous protists in the rhizosphere on maize plant growth parameters and the bacterial community composition. Beyond that we investigated the induction of plant biochemical responses by separately analyzing above- and below-ground metabolite profiles of maize plants incubated either with a soil bacterial inoculum or with a mixture of soil bacteria and bacterivorous protists. Significantly distinct leaf and root metabolite profiles were obtained from plants which grew in the presence of protists. These profiles showed decreased levels of a considerable number of metabolites typical for the plant stress reaction, such as polyols, a number of carbohydrates and metabolites connected to phenolic metabolism. We assume that this decrease in plant stress is connected to the grazing induced shifts in rhizosphere bacterial communities as shown by distinct T-RFLP community profiles. Protist grazing had a clear effect on the overall bacterial community composition, richness and evenness in our microcosms. Given the competition of plant resource allocation to either defense or growth, we propose that a reduction in plant stress levels caused directly or indirectly by protists may be an additional reason for corresponding positive effects on plant growth.

Renewable electrical energy production, such as wind power or photovoltaics, faces temporal fluctuations and spatial separation of source and sink creating the need for suitable storage and conversion technologies. Furthermore, a bio-based economy needs technologies allowing the on-demand and on-site conversion of biomass to platform chemicals and chemical energy carriers. This study combines microbial and electrochemical conversions, allowing production of chemicals from biomass and electricity. Carboxylic acids (CAs) are produced by anaerobic conversion of a renewable feedstock with reactor microbiomes. CAs are subsequently electrochemically upgraded to yield energy-dense alkanes. We present a proof-of-concept based on experimental data for each step in two independent case studies at laboratory scale. During continuous CA fermentation from corn beer, mainly n-caproic and n-caprylic acid were produced yielding in total 0.638 g COD g−1 COD [CA/corn beer]. A batch system fed with corn silage yielded mainly n-butyric acid (0.207 g COD g−1 COD [CA/corn silage]), and some n-caproic acid (0.030 g COD g−1 COD [CA/corn silage]). The CAs were removed from the fermentation broth using a pertraction system. Subsequently, the CAs were electrochemically converted via Kolbe reaction and other reaction pathways. Depending on the carbon chain length of the CAs, liquid alkanes (from medium-chain CAs) or gaseous alkanes and alcohols (from short-chain CAs) were gained. Coulombic efficiencies of up to 80% were achieved despite the alkaline (pH 9) electrolysis conditions that are dictated by the pertraction system. Remarkably, CAs with a carbon chain length of six or more C-atoms were successfully converted to liquid alkanes at a high yield (in total: 0.480 g COD g−1 COD [hydrocarbon/corn beer]). This was achieved at an energy input of only 0.100 kW h per mol of converted CA (considering the electrochemical half cell reaction). Noteworthily, the electrochemical CA conversion is much f

Our experiments were focused on the metabolic footprint of mineral-nutrient availability under field conditions. While there are multiple factors potentially blurring such footprints, we hypothesized that physiological and metabolic adaptations of established plants are particularly important mechanisms under field conditions. To study respective differences between young and established plants and to study the impact of disturbances on the adaptive capacity of established plants, we analyzed Medicago sativa plants of different age from plots with marked differences in the levels of soil mineral nutrients established in a long-term fertilization experiment. Gas chromatography–mass spectrometry was used to determine metabolite profiles of sink and source leaves of plants in an early state of development (“young plants”), just before the first harvest (“established plants”), and a short time after the second harvest (“regrowing plants”). Metabolite profiles from young plants were markedly responsive to soil mineral nutrients and resembled respective profiles from controlled conditions, demonstrating that overall variability of growth and sampling conditions had relatively little importance for the metabolite profiles recorded. In the case of established plants, however, we observed only little impact of availability of mineral nutrients on metabolite profiles. This low metabolic responsiveness of plants was partially lost after severe disturbances (removal of the plant shoot). Metabolite profiling, in summary, is able to detect a metabolic footprint of mineral-nutrient availability in young plants under field conditions and may provide information about the ability of older plants to partially uncouple their metabolism from the environment. In addition, it is also possible to determine the impact of disturbances on this ability of the plant organism.

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.

Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.

Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A calibration procedure based on Principal Component Regression was developed for estimating the ratio of the bacterial species. Data for method calibration were gained from pure cultures and artificially assembled communities of known ratios of the two member populations. Moreover, to account for physiological variability, FT-IR measurements were performed with organisms sampled at different growth phases. Measurements and data analyses were subsequently applied to growing mixed cultures revealing that growth of R. ruber was almost completely suppressed in co-culture with P. putida. Population ratios obtained by fatty acid analysis as an independent reference method were in high agreement with the FT-IR derived ratios.

Electrokinetically stimulated bioremediation of soils (electro-bioremediation) requires that the application of weak electric fields has no negative effect on the contaminant degrading microbial communities. This study evaluated the hypothesis that weak direct electric current (DC) fields per se do not negatively influence the physiology and composition of soil microbial communities given that secondary electrokinetic phenomena such as soil pH changes and temperatures are minimized. Mildly buffered, water-saturated laboratory mesocosms with agricultural soil were subjected for 34 days to a constant electric field (X = 1.4 V cm- 1; J 1.0 mA cm- 2) and the spatiotemporal changes of soil microbial communities assessed by fingerprints of phospholipids fatty acids (PLFA) and terminal restriction fragment length polymorphisms (T-RFLP) of bacterial 16S rRNA genes. DC-induced electrolysis of the pore water led to pH changes (< 1.5 pH units) in the immediate vicinity of the electrodes and concomitant distinct soil microbial community changes. By contrast, DC-treated bulk soil distant to the electrodes showed no pH changes and developed similar PLFA- and T-RFLP-fingerprints as control soil in the absence of DC. Our data suggest that the presence of an electric field, if suitably applied, will not influence the composition and physiology of soil microbial communities and hence not affect their potential to biodegrade contaminants.

The epidermis of plants is coated by a thin cuticular membrane protecting the plant from water loss and UV radiation. This lipid layer includes long-chained fatty acids, esters, hydrocarbons, alcohols, aldehydes and ketones. The composition of these compounds depends on the species, their ages and growing conditions. Also chemical stress seems to change the qualitative and quantitative composition of the lipid layers caused by pollutant interfered plants biosynthesis. Particularly the chain length, saturation and branching of fatty acids change during pollutant exposure as previous studies indicated. In this project especially the wax layer of juncus effusus and phragmitis communis were studied under chemical stress caused by chlorobenzene and hydrocarbon contaminated water. The extractable surface lipids were isolated from the plants and analyzed by gas chromatography-mass spectrometry after derivatization. For data processing a pattern recognition program was used that based on a costume made data base containing typical plant lipid substances. First results indicate that in comparison to the control plants the composition and particularly the amount of the wax components changed clearly in the exposed plants. Statistical evaluation of the data confirmed the alterations determined as significant influence of the pollutants.

A multidimensional approach for the identification of fatty acid methyl esters (FAME) based on GC/MS analysis is described. Mass spectra and retention data of more than 130 FAME from various sources (chain lengths in the range from 4 to 24 carbon atoms) were collected in a database. Hints for the interpretation of FAME mass spectra are given and relevant diagnostic marker ions are deduced indicating specific groups of fatty acids. To verify the identity of single species and to ensure an optimized chromatographic resolution, the database was compiled with retention data libraries acquired on columns of different polarity (HP-5, DB-23, and HP-88). For a combined use of mass spectra and retention data standardized methods of measurement for each of these columns are required. Such master methods were developed and always applied under the conditions of retention time locking (RTL) which allowed an excellent reproducibility and comparability of absolute retention times. Moreover, as a relative retention index system, equivalent chain lengths (ECL) of FAME were determined by linear interpolation. To compare and to predict ECL values by means of structural features, fractional chain lengths (FCL) were calculated and fitted as well. As shown in an example, the use of retention data and mass spectral information together in a database search leads to an improved and reliable identification of FAME (including positional and geometrical isomers) without further derivatizations.

Strains L10T, L108 and CIP I-2052 were originally obtained from methyl tert-butyl ether (MTBE)-contaminated groundwater and from a wastewater treatment plant, respectively. All share the ability to grow on tert-butanol, an intermediate of MTBE degradation. Cells are strictly aerobic, motile by a polar flagellum and exhibit strong pili formation. Poly -hydroxybutyrate (PHB) granules are formed. The DNA G+C content is 69-70.5 mol% and the main ubiquinone is Q-8. The major cellular fatty acids are 16 : 1 cis-9 and 16 : 0 and the only hydroxy fatty acid is 10 : 0 3-OH. The major phospholipids are phosphatidylethanolamine (PE) 16 : 1/16 : 1 and phosphatidylglycerol 16 : 0/16 : 1. A significant amount of PE 17 : 0/16 : 1 is present. The 16S rRNA gene sequences of these strains are almost identical and form a separate line of descent in the Rubrivivax-Roseateles-Leptothrix-Ideonella-Aquabacterium branch of the Betaproteobacteria with 97 % similarity to 16S rRNA genes of the type strains of Rubrivivax gelatinosus, Leptothrixmobilis and Ideonella dechloratans. However, physiological properties, DNA-DNA relatedness values and the phospholipid and cellular fatty acid profiles distinguish the novel isolates from the three closely related genera. Therefore, it is concluded that strains L10T, L108 and CIP I-2052 represent a new genus and novel species for which the name Aquincola tertiaricarbonis gen. nov., sp. nov., is proposed. The type strain is strain L10T (=DSM 18512T=CIP 109243T).

The impact of cis, trans and cyclopropane fatty acids on membrane fluidity was investigated using batch-grown Pseudomonas putida P8 and Comamonas testosteroni ATCC 17454. A major difference observed between the two investigated strains is the absence of the ability to synthesize trans-unsaturated fatty acids in Comamonas. When grown exponentially at 30 °C, a shift to 35 °C increased the trans/cis ratios of the fatty acids of P. putida P8 from 0 to 0.81 and 0 to 1.07, in lipid extracts and cell hydrolyzates, respectively. After prolonged growth followed by nutrient deprivation for 48 h, both at 30 °C, trans fatty acids were absent, but the cyclo/cis ratios rose from 0.1 to 1.55 in lipid extracts, and from 0.1 to 1.54 in cell hydrolyzates. C. testosteroni ATCC 17454 contained no cyclo fatty acids when harvested in the exponential phase after 6 h, whereas after 72 h cultivation, the cyclo/cis ratios rose to 0.49 and 0.47, in lipid extracts and cell hydrolyzates, respectively. Trans fatty acids were never observed in this strain. Increased cyclo/cis and trans/cis ratios correlated with decreased fluidity measured by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) intercalated in the bilayers of liposomes and by Fourier Transform Infrared (FTIR) spectroscopy of lipids prepared from the cells. The specific effect of cyclopropane fatty acids on membrane fluidity was much smaller than that of trans fatty acids. FTIR-measurements of intact cells of P. putida P8 confirmed the high potency of trans fatty acids to decrease the fluidity. In cells with induced cyclopropane fatty acid synthesis, the membranes remained more fluidized, indicating the lower importance of these fatty acids for homeoviscosis.

Fatty acid compositions in growing and resting cells of several strains of Pseudomonas putida (P8, NCTC 10936, and KT 2440) were studied, with a focus on alterations of the saturation degree, cis-trans isomerization, and cyclopropane formation. The fatty acid compositions of the strains were very similar under comparable growth conditions, but surprisingly, and contrary to earlier reports, trans fatty acids were not found in either exponentially growing cells or stationary-phase cells. During the transition from growth to the starvation state, cyclopropane fatty acids were preferentially formed, an increase in the saturation degree of fatty acids was observed, and larger amounts of hydroxy fatty acids were detected. A lowered saturation degree and concomitant higher membrane fluidity seemed to be optimal for substrate uptake and growth. The incubation of cells under nongrowth conditions rapidly led to the formation of trans fatty acids. We show that harvesting and sample preparation for analysis could provoke the enzyme-catalyzed formation of trans fatty acids. Freeze-thawing of resting cells and increased temperatures accelerated the formation of trans fatty acids. We demonstrate that cis-trans isomerization only occurred in cells that were subjected to an abrupt disturbance without having the possibility of adapting to the changed conditions by the de novo synthesis of fatty acids. The cis-trans isomerization reaction was in competition with the cis-to-cyclopropane fatty acid conversion. The potential for the formation of trans fatty acids depended on the cyclopropane content that was already present.

This study explored the capability of Pseudomonas putida NCTC 10936 to maintain homeoviscosity after changing the growth temperature, incubating resting cells at different temperatures or at a constant temperature in the presence of 4-chlorophenol (4-CP). After raising the growth temperature from 20 to either 30 or 35°C, the degree of saturation of the organism’s fatty acids increased and the ratio of trans to cis unsaturated fatty acids decreased somewhat. In contrast, after the incubation temperature of resting cells was raised (grown at 30°C) from 20 to 30 or 35°C the degree of saturation of the fatty acids remained nearly constant, while the ratio of trans to cis unsaturated fatty acids increased. Incubating resting cells (grown at 30°C) at 20°C in the presence of 4-CP again caused no major changes in the degree of saturation, but cis to trans conversion of unsaturated fatty acids was induced, with a corresponding increase in the trans/cis ratios. Increases in both the saturation degree of the fatty acids and the trans/cis ratio of the unsaturated fatty acids correlated with increases in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene intercalated in the bilayers of liposomes prepared from the cells of P. putida NCTC 10936. Electron transport phosphorylation (ETP) could be stabilized by adaptive adjustments in the fluidity of the cytoplasmic membrane mediated by changes in fatty acid composition such as those observed. Whether changes in the degree of saturation or in the trans/cis ratio are more effective can be decided by studying P. putida NCTC 10936.

Bacteria often use pollutants as sole carbon and energy sources. However, if they are toxic and the concentrations are high these compounds inhibit growth, and eventually poison the biocatalysts. In order to identify mechanisms contributing to stability, the protein patterns of Acinetobacter calcoaceticus during growth on potentially toxic phenol were analysed using two-dimensional gel electrophoresis. Enzymes involved in the catabolism or assimilation of phenol, such as phenol monooxygenase and catechol 1,2-dioxygenase, were induced at a more than twofold level in response to long-term exposure to high concentrations of the compound serving as the sole carbon and energy source. This would have clear adaptive benefits, since increased rates of consumption of phenol would reduce the susceptibility of Acinetobacter calcoaceticus to phenol poisoning. In contrast, transient induction of only one heat shock protein and one oxidative stress protein was detected during long-term exposure to high concentrations of phenol.

RESUMEN Las bacterias responden a los cambios ambientales modificando su composición, para evitar el daño que dichos cambios podrían ejercer. Una de las modificaciones más importantes es la variación de la composición de los ácidos grasos de las membranas celulares, que le permite mantener la homeoviscosidad ante situaciones de estrés. Trabajos previos han estudiado la acción de la temperatura, presión hidrostática y diferentes solventes sobre cepas de Pseudomonas putida. En este trabajo se estudió la acción conjunta de la temperatura y la salinidad sobre la composición de ácidos grasos de membranas celulares de Pseudomonas fluorescens GNP-OHP-3, una cepa bacteriana aislada de un hábitat contaminado con petróleo. Pseudomonas fluorescens GNP-OHP-3 respondió a las variaciones de temperatura modificando los ácidos grasos de sus membranas de manera similar a lo descripto en otros integrantes de su género: ante el aumento de temperatura se observó un incremento de ácidos grasos saturados y una disminución de los ácidos grasos insaturados. En el rango de concentraciones salinas ensayadas las variaciones de los ácidos grasos mayoritarios fueron en general erráticas. La respuesta de los ácidos grasos ciclo propano pudo expresarse con ecuaciones matemáticas que permitieron predecir el porcentaje de estos ácidos en relación a la concentración de cloruro de sodio. SUMMARY Influence of salinity and temperature on fatty acid composition of Pseudomonas fluorescens GNP-OHP-3 membrane. The bacteria respond to environmental changes modifying their composition. One of the most important modifications is the variation on fatty acid composition of cellular membranes to maintain the homeoviscosity. The action of temperature, hydrostatic pressure and solvents on Pseudomonas putida has been thoroughly studied. In this paper, the combined action of the temperature and salinity on fatty acid composition of cellular membranes of Pseudomonas fluorescens GNP-OHP-3, a bacterial strain isolated

The toxicity of chlorophenoxy herbicides to a bacterium. strongly related to the well-known species Delftia (formerly Comamonas) acidovorans that are able to detoxify these xenobiotics, was investigated. The oxidation of n-hexanol via alcohol dehydrogenases, coupled with the generation of ATP by electron transport phosphorylation (ETP), was used as an indicator for energy-toxic effects on the growth of Comamonas testosteroni ATCC 17454. Uncoupling-reductions in ATP synthesis accompanied by increased respiration-was found to be induced by 1 mM of the classic uncoupler 2,4-dinitrophenol (2,4-DNP) at pH 7.0 and 8.0. At pH 5.4 and 6.0, the ATP synthesis and respiration were strongly inhibited by both 2,4-DNP and the chlorophenoxy herbicides tested. In contrast, 5 mM of 2,4-dichlorophenoxyacetic acid (2,4-D) and of 2-(2,4-dichlorophenoxy)-propanoic acid (2,4-DCPP) were required for detectable uncoupling effects-reduction of the P/O ratios by about 30%-at pH 7.0. These chemicals may have less uncoupling power because the concentration of their protonated (undissociated) forms (pKa values 2.7 and 3.0) is an order of magnitude lower than that of 2,4-DNP (pKa = 4.0) at this pH value. Strong uncoupling accompanied by increased respiration. like that induced by 1 mM 2,4-DNP, was also caused by 5 mM 4-(2,4-dichlorophenoxy)-butyric acid (2,4-DCPB), which correlates with its high pKa value of 4.6. The order of toxicity of the chlorophenoxy herbicides (2,4-D < 2,4-DCPP < 2,4-DCPB) to the ETP, which correlates well with the lipophilicity of their undissociated forms (log P 2.7 < 3.4 < 3.5, respectively), was confirmed by measuring their capacity to inhibit the growth of Comamonas testosteroni ATCC 17454. The results show that energization via alcohol dehydrogenases can be used as an indicator for investigating energy-toxic effects of organics on the ETP and growth of chlorophenoxy herbicide-detoxifying bacteria.

This study explored the capability of Acinetobacter calcoaceticus 69V for homeoviscous adaptation. The saturation degree of the organism's fatty acids increased as the growth temperature was raised. This response was correlated with an increase in the fluorescence anisotropy of 1,6-diphenyl- 1,3,5-hexatriene (DPH) intercalated in the bilayers of liposomes prepared from the cells, indicating that the fluidity of their outer and cytoplasmic membranes fell as growth temperature increased. The liposomes' fluidity increased as the temperature at which it was measured was raised. The homeoviscous efficacy of the responses to increases in growth temperature was 0.3 to 0.5, according to differences between the temperature profiles of cells grown at 20 and 40 °C. However, the derived figure fell to zero at 42 °C, the maximum growth temperature of this bacterium. Similar differences in the temperature profiles of fluorescence anisotropy were obtained from liposomes with differing degrees of saturation in their fatty acids induced by growing the organism on various concentrations of the lipophilic substrates ethanol and phenol. Adjusting the saturation degree of the fatty acids seems to play a major role in the homeostasis of viscosity of the cytoplasmic membrane of Acinetobacter calcoaceticus 69V, a feature that is critical for stabilising its apparatus for electron transport phosphorylation (ETP) against lipophilic attack by xenobiotics.

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This study explored the suitability of using the trans/cis ratio of unsaturated fatty acids as an indicator of the acute toxicity of membrane active hazardous chemicals. The conversion of cis into trans fatty acids in Pseudomonas putida NCTC 10936 in response to 4-chlorophenol and temperature changes was compared with the results from another kind of toxicity test using the same organism, based on the sensitivity of its xylose oxidation-driven ATP synthesis to uncoupling. The response of both indicators is believed to be largely due to changes in the fluidity of the cytoplasmic membrane. However, the electron transport phosphorylation reacted faster and more sensitively to the fluidizing effect of 4-chlorophenol than the isomerization of unsaturated fatty acids. Therefore, measuring the trans/cis ratio does not provide as good early warning signals of acute toxicity as monitoring the response of the electron transport phosphorylation. If used as an indicator of chemostress, with Pseudomonas species as test organisms, the ratio should only be used in conjunction with other parameters reflecting the energetic state of the cells.

Autochthonous bacteriocenoses in semiarid soils in Patagonia were found to be capable of rapidly adapting to high contamination with crude oil. This adaptation at community level is due to the selective enrichment of hydrocarbon-utilizing bacteria always present in these soils. Immediately after a heavy contamination with crude oil, the authochthonous bacteriocenosis contained about 28% hydrocarbon-utilizing bacteria which could be classified into eight ecotypes with characteristic metabolic profiles. Mainly n-alkanes were used as growth substrates of representative strains. After seven months' exposure to crude oil, the bacteriocenosis consisted almost entirely of hydrocarbon-utilizing bacteria. At least fourteen ecotypes were distinguishable, and the majority of representative strains were able to metabolize a broad spectrum of aliphatic and aromatic hydrocarbons. Corresponding to the significant alteration of the physiological diversity, drastic changes to the taxonomic diversity were also found. Whereas at the beginning of the study the autochthonous bacteriocenoses were dominated by GRAM-positive genera of the Actinomycetales (Dietzia, Gordona, Nocardia, Rhodococcus, Streptomyces) with high ecological potency, after just two months' exposure to crude oil, GRAM- negative bacteria (especially Pseudomonas stutzeri) became predominant within the hydrocarbon-utilizing bacteriocenoses accompanied by some GRAM-positive genera of the Actinomycetales with a significantly lower abundance. These findings underline the importance of Pseudomonas and some genera of Actinomycetales for processes of natural attenuation and the technically supported in situ bioremediation of soil polluted by crude oil in Patagonia.

Taxonomic diversity and the hydrocarbon-degrading potential of heterotrophic, aerobic biocenoses of three different ecosystems (mineral oil-contaminated, unpolluted and bioremediated soils) were assessed by characterizing and identifying their main bacterial constituents. All colonies grown on universal (R2A agar) and selective (mineral agar with different mineral oils as the sole C-source) media on plates with suitable colony densities were isolated. The automated identification systems BIOLOG and MIS/SHERLOCK were used for the rapid identification of these isolates. In each case, the assignment to a taxon was verified by determining further diagnostically relevant parameters (cell and colony morphology, selected biochemical reactions, chemotaxonomical criteria). Corresponding to earlier results, the BIOLOG system was found to provide satisfactory results in the identification of Gram- bacteria whereas within the group of Gram+ isolates only a few taxa were identified reliably. MIS/SHERLOCK provided sufficient results for a wide range both of Gram- and Gram+ isolates. The occurrence of characteristic fatty acids even allowed in some cases reliable taxonomical assignment to a genus despite relatively low similarity indices. However, some apparently reliable identification results obtained with both identification systems proved to be incorrect according to additional diagnostic methods applied. The combined use of BIOLOG and MIS/SHERLOCK in connection with some easily accessible and routinely determined diagnostically relevant properties resulted in rates of correct identification of approximately 75% at genus level and and about 30 to 60% at species level (compared to the total number of isolates). Because of the very different identification results within certain taxonomical and physiological groups, the percentage of identifiable bacterial components of terrestrial biocenoses varied with their taxonomic composition.

In the detoxifying bacteriumAcinetobacter calcoaceticus69-V and in the acetic acid bacteriumAcetobacter methanolicusMB 58, glucose and xylose are oxidized, respectively, via PQQ-dependent membrane-bound dehydrogenases, which are linked to the respiratory chain in a manner enabling energy conservation via electron transport phosphorylation (ETP) in the cytoplasmic membrane. Neither the glucose and gluconic acid nor the xylose and xylonic acid are metabolized. Therefore, measurements of sugar oxidation-driven ATP syntheses ought not to be disturbed by ATP drainage caused by anabolic processes. Studying the effect of substituted phenolic compounds on these energization processes reveals that their toxicity increases with an increasing degree of chlorination and thatA. calcoaceticus69-V is more stable thanA. methanolicusMB 58 against chlorinated phenols. On the other hand,A. methanolicusMB 58 is more stable against 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D), especially in the acidic pH range, in which the sensitivity of ATP synthesis to the uncouplers is higher than that of respiration. The toxicity caused by protonophoric activities ought to be barely detectable by respiratory and dehydrogenase tests. The luminescence system ofPhotobacterium phosphoreumtested in the luminescent bacteria test was much more sensitive. This test system should be used as a screening tool and the effects measured must be confirmed by toxicity tests evaluating the stability of bacteria themselves involved in processes of detoxification as well as the production of toxic metabolites, monitored with respect to their velocity and efficiency.