Maintenance and impacts of an inoculated mer/luc-tagged Pseudomonas fluorescens on microbial communities in birch rhizospheres developed on humus and peat.

Antagonistic bacteria represent promising biocontrol agents for improving forest production in seedling nurseries or forest soils. The fate of an introduced mer/luc-tagged antagonistic Pseudomonas fluorescens 31K3 was monitored in the rhizosphere of silver birch (Betula pendula) seedlings grown in microcosms containing forest humus or nursery peat. The inoculated strain (10(8) cfu g(-1) soil) was unable to establish in significant numbers in either soil type and turned nonculturable in humus. Detection in both soils was possible only via luminescence of enrichment cultures 80 days post-inoculation. Despite low P. fluorescens survival, inoculation had a positive effect on seedling growth. Limited impact of inoculation on the indigenous microbial communities was identified following analyses of respiration and denitrification potential, community-level physiological profiles and molecular fingerprinting of fungi and eubacteria, and Pseudomonas community structures. The minor changes observed in the indigenous microbial communities, including mycorrhiza development, were not consistent between humus and peat growth substrates. It was concluded that the rhizosphere-related microbial communities developed in both of these highly organic soil systems are highly buffered against introduction of foreign bacteria.

Microcosm-based analyses of Scots pine seedling growth, ectomycorrhizal fungal community structure and bacterial carbon utilization profiles in boreal forest humus and underlying illuvial mineral horizons.

We report on the identity of indigenous mycorrhiza forming fungi and rhizosphere/mycorrhizosphere bacterial community carbon source utilization profiles of Scots pine (Pinus sylvestris L.) seedlings grown in boreal forest humus (O) or illuvial (B) mineral horizon containing microcosm growth systems. Based on rDNA (ITS)-RFLP analyses, a total of 10 fungal RFLP taxa were identified from pre-morphotyped mycorrhizas on 7-month-old seedling roots. Hierarchical cluster analysis, including corresponding RFLPs of known fungal species, confirmed root colonization by eight mycorrhizal species. In the O horizon, roots were colonized by e.g. Suillus bovinus, Suillus variegatus, Cenococcum geophilum, Piloderma croceum, Thelephora terrestris and Russula vinicolor. Mycobiont diversity in the mineral B horizon was lower but included Piceirhiza bicolorata and both Suillus species which produced extensive extramatrical mycelium. In comparison to non-colonized soils, rhizosphere and mycorrhizosphere compartments supported significantly higher numbers of bacteria (mean range 10(8)-10(11) cells g(-1) fresh weight (fw)). Specific rhizosphere/mycorrhizosphere 'niche'-linked bacterial communities were detected following multivariate analyses (PCA and CA) of bacterial carbon utilization profiles (Biolog(R) GN microplate). Distinct preferences for amino and carboxylic acids were identified in mineral B horizon rhizospheres whereas a wider range of carbon sources were utilized in the fungal-dominated mycorrhizospheres irrespective of soil types.

Effects of Pinus sylvestris root growth and mycorrhizosphere development on bacterial carbon source utilization and hydrocarbon oxidation in forest and petroleum-contaminated soils.

The hypothesis that Pinus sylvestris L. root and mycorrhizosphere development positively influences bacterial community-linked carbon source utilization, and drives a concomitant reduction in mineral oil levels in a petroleum hydrocarbon- (PHC-) contaminated soil was confirmed in a forest ecosystem-based phytoremediation simulation. Seedlings were grown for 9 months in large petri dish microcosms containing either forest humus or humus amended with cores of PHC-contaminated soil. Except for increased root biomass in the humus/PHC treatment, there were no other significant treatment-related differences in plant growth and needle C and N status. Total cell and culturable bacterial (CFU) densities significantly increased in both rhizospheres and mycorrhizospheres that actively developed in the humus and PHC-contaminated soil. Mycorrhizospheres (mycorrhizas and extramatrical mycelium) supported the highest numbers of bacteria. Multivariate analyses of bacterial community carbon source utilization profiles (Biolog GN microplate) from different rhizosphere, mycorrhizosphere, and bulk soil compartments, involving principal component and correspondence analysis, highlighted three main niche-related groupings. The respective clusters identified contained bacterial communities from (i) unplanted bulk soils, (ii) planted bulk PHC and rhizospheres in PHC-contaminated soils, and (iii) planted bulk humus and rhizosphere/mycorrhizosphere-influenced humus, and mycorrhizosphere-influenced PHC contaminated soil. Correspondence analysis allowed further identification of amino acid preferences and increased carboxylic/organic acid preferences in rhizosphere and mycorrhizosphere compartments. Decreased levels of mineral oil (non-polar hydrocarbons) were detected in the PHC-contaminated soil colonized by pine roots and mycorrhizal fungi. These data further support our view that mycorrhizosphere development and function plays a central role in controlling associated bacterial communities and their degradative activities in lignin-rich forest humus and PHC-contaminated soils.

Biomarkers for monitoring efficacy of bioremediation by microbial inoculants.

Bioaugmentation of contaminated sites with microbes that are adapted or genetically engineered for degradation of specific toxic compounds is an area that is currently being explored as a clean-up option. Biomarkers have been developed to track the survival and efficacy of specific bacteria that are used as inocula for bioremediation of contaminated soil. Examples of biomarkers include the luc gene, encoding firefly luciferase and the gfp gene, encoding the green fluorescent protein (GFP). The luc gene was used to tag different bacteria used for bioremediation of gasoline or chlorophenols. The bacteria were monitored on the basis of luciferase activity in cell extracts from soil. The gfp gene was also used to monitor bacteria during degradation of chlorophenol in soil, based on fluorescence of the GFP protein. Other biomarkers can also be used for monitoring of microbial inocula used for bioaugmentation of contaminated sites. The choice of biomarker and monitoring system depends on the particular site, bacterial strain and sensitivity and specificity of detection required.

Bioremediation of petroleum hydrocarbon-contaminated soil by composting in biopiles.

Composting of contaminated soil in biopiles is an ex situ technology, where organic matter such as bark chips are added to contaminated soil as a bulking agent. Composting of lubricating oil-contaminated soil was performed in field scale ( [Formula: see text] m(3)) using bark chips as the bulking agent, and two commercially available mixed microbial inocula as well as the effect of the level of added nutrients (N,P,K) were tested. Composting of diesel oil-contaminated soil was also performed at one level of nutrient addition and with no inoculum. The mineral oil degradation rate was most rapid during the first months, and it followed a typical first order degradation curve. During 5 months, composting of the mineral oil decreased in all piles with lubrication oil from approximately 2400 to 700 mg (kg dry w)(-1), which was about 70% of the mineral oil content. Correspondingly, the mineral oil content in the pile with diesel oil-contaminated soil decreased with 71% from 700 to 200 mg (kg dry w)(-1). In this type of treatment with addition of a large amount of organic matter, the general microbial activity as measured by soil respiration was enhanced and no particular effect of added inocula was observed.

Straw compost and bioremediated soil as inocula for the bioremediation of chlorophenol-contaminated soil.

We evaluated the use of straw compost and remediated soil as inocula for bioremediation of chlorophenol-contaminated soil. The in situ biotransformation of pentachlorophenol (PCP) and mineralization of radiolabeled [U-(sup14)C]PCP by straw compost and remediated soil were studied under field-simulating conditions before and after 3 months of adaptation with PCP in a percolator. After PCP adaptation, the straw compost mineralized up to 56% of the [(sup14)C]PCP. No partial dechlorination of PCP was found. The native straw compost did not mineralize PCP, but partial dechlorination of PCP occurred (i) at pH 8 under near-thermophilic conditions (45(deg)C) and (ii) at pH 7 under aerobic and mesophilic conditions. No biotransformation reactions occurred at room temperature (25(deg)C) at pH 8. Enrichment in the percolator enhanced the mineralization rate of remediated soil to 56% compared with that of the native remediated soil, which mineralized 24% of [(sup14)C]PCP added. Trace amounts of chloroanisoles as the only biotransformation products were detected in PCP-adapted remediated soil. Both inoculants studied here showed effective mineralization of PCP when they were adapted to PCP in the percolator. No harmful side reactions, such as extensive methylation, were observed.

Polyphosphate accumulation among denitrifying bacteria in activated sludge.

Bacterial polyphosphate accumulation and denitrification are important processes in biological removal of nutrients from wastewater. It has been suggested that phosphorus accumulators are able to denitrify. However, the bacteria known as the most important phosphorus accumulators, belonging to the genus Acinetobacter are generally not known to denitrify. To clarify how commonly both physiological traits are present in the same organism, we screened 165 isolates from activated sludge and wastewater for their ability to denitrify, and the ability of the denitrifying isolates to accumulate polyphosphate. Of the 165 isolates, 149 were from acetate mineral medium (87 of these identified as Acinetobacter by the API 20 NE identification system) and 16 were from nutrient broth and nitrate medium. Only 15 of 165 isolates tested showed true respiratory denitrification activity. In the presence of acetylene they converted more than 80% of 5mM NO3- to N2O in 6 days. None of the Acinetobacter isolates were among the 15 respiratory denitrifiers. The denitrifying isolates were identified as species of Pseudomonas, Agrobacterium, Pasteurella, Sphingomonas or could not be identified by the API 20 NE identification system. According to the BIOLOG identification system the denitrifiers were species of Pseudomonas, Hydrogenophaga, Citrobacter, Xanthomonas or they could not be identified. The ability of confirmed denitrifiers to accumulate phosphate was measured in experiments where cells pregrown under phosphorus limitation were exposed to phosphate (8 mg P/L) under aerobic conditions. The rates of excess phosphate uptake varied from 0.3 to more than 23 mg P/g dry matter/h. Rates for four isolates were higher than those reported for Acinetobacter strains. These results show that polyphosphate accumulation and denitrification in activated sludge can be carried out by the same organisms.

Survival of denitrifiers in nitrate-free, anaerobic environments.

Experiments were undertaken to explain the occurrence of a high denitrification capacity in anaerobic, NO(3)-free habitats. Deep layers of freshwater sediments that were buried more than 40 years ago and digested sludge were the habitats studied. The denitrifier populations were 3.1 x 10 and 3.1 x 10 cells cm in deep sediments from a river and lake, respectively, and 5.3 x 10 cells cm in digested sludge. The denitrification capacities of the samples reflected the population densities. Strict anaerobic procedures were used to obtain the predominant isolates that would grow on anaerobic medium with NO(3). All strict anaerobes isolated failed to denitrify. All isolates that denitrified were aerobic, gram-negative bacteria, particularly species of Pseudomonas and Alcaligenes. No detectable growth was observed when these strains were incubated with electron acceptors other than NO(3) or O(2). When representative isolates were added to sterile, O(2)- and NO(3)-free porewater from their original locations at their natural densities (10 cells cm), no change in viable population was noted over 3 months of incubation. Metabolic activity was demonstrated in these cells by slow formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [C]glucose was added to cell suspensions of the pseudomonads that had been starved for 3 months without electron acceptors (O(2) or NO(3)), C-labeled products, including cell biomass, CO(2), and fermentation products, were produced. The high denitrification capacity of these anaerobic environments appears to be due to conventional respiratory denitrifiers. These organisms have the capacity for long-term survival without O(2) or NO(3) and appear to be capable of providing for their maintenance by carrying on a low level of fermentation.

Annual pattern of denitrification and nitrate ammonification in estuarine sediment.

The seasonal variation and depth distribution of the capacity for denitrification and dissimilatory NO(3) reduction to NH(4) (NO(3) ammonification) were studied in the upper 4 cm of the sediment of Norsminde Fjord estuary, Denmark. A combination of C(2)H(2) inhibition and N isotope techniques was used in intact sediment cores in short-term incubations (maximum, 4 h). The denitrification capacity exhibited two maxima, one in the spring and one in the fall, whereas the capacity for NO(3) ammonification was maximal in the late summer, when sediments were progressively reduced. The denitrification capacity was always highest in the uppermost 1 cm of the sediment and declined with depth. The NO(3) ammonification was usually higher with depth, but the maximum activity in late summer was observed within the upper 1 cm. The capacity for NO(3) incorporation into organic material was investigated on two occasions in intact sediment cores and accounted for less than 5% of the total NO(3) reduction. Denitrification accounted for between 13 and 51% of the total NO(3) reduction, and NH(4) production accounted for between 4 and 21%, depending on initial rates during the time courses. Changes of the rates during the incubation were observed in the late summer, which reflected synthesis of denitrifying enzymes. This time lag was eliminated in experiments with mixed sediment because of preincubation with NO(3) and alterations of the near-environmental conditions. The initial rates obtained in intact sediment cores therefore reflect the preexisting enzyme content of the sediment.