Link between spatial structure of microbial communities and degradation of a complex mixture of volatile organic compounds in peat biofilters.

AIMS: To investigate the relationships between the operation of the volatile organic compound (VOC) removal biofilter and the structure of microbial communities, and to study the impact on degradation activities and the structuring of microbial communities of biofilter malfunctions related to the qualitative composition of the polluted air. METHODS AND RESULTS: A microbiological study and a measurement of biodegradation activities were simultaneously carried out on two identical peat-packed columns, seeded with two different inocula, treating polluted air containing 11 VOCs. For both reactors, the spatial structure of the microbial communities was investigated by means of single-strand conformation polymorphism (SSCP) analysis. For both reactors, stratification of degradation activities in function of depth was observed. Oxygenated compounds were removed at the top of the column and aromatics at the bottom. Comparison of SSCP patterns clearly showed a shift in community structure in function of depth inside both biofilters. This distribution of biodegradation activities correlates with the spatialization of microbial density and diversity. Although the operating conditions of both reactors were identical and the biodegradation activities similar, the composition of microflora differed for biofilters A and B. Subdivision of biofilter B into two independent parts supplied with polluted air containing the complex VOC mixture showed that the microflora having colonized the bottom of biofilter B retained their potential for degrading oxygenated compounds. CONCLUSIONS: This work highlights the spatialization of biodegradation functions in a biofilter treating a complex mixture of VOCs. This distribution of biodegradation activities correlates with the spatialization of microbial density and diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This vertical structure of microbial communities must be taken into consideration when dealing with the malfunctioning of bioreactors. These results are also useful information about changes in microbial communities following natural or anthropogenic alterations in different ecosystems (soils and sediments) where structuring of microbial communities according to depth has been observed.

Immunological method for direct assessment of the functionality of a denitrifying strain of Pseudomonas fluorescens in soil.

This work describes an immunological method for detection and quantification in complex environments of the dissimilative nitrate reductase (NRA) responsible for the reduction of nitrate to nitrite, which plays an important role in ecosystem functioning. The alpha-catalytic subunit of the enzyme was purified from the denitrifying strain Pseudomonas fluorescens YT101 and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NRA by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. The specificity, detection threshold and reproducibility of the proposed method were evaluated. A soil experiment showed that our method can be applied to complex environmental samples.

Role of respiratory nitrate reductase in ability of Pseudomonas fluorescens YT101 to colonize the rhizosphere of maize.

Selection of the denitrifying community by plant roots (i.e., increase in the denitrifier/total heterotroph ratio in the rhizosphere) has been reported by several authors. However, very few studies to evaluate the role of the denitrifying function itself in the selection of microorganisms in the rhizosphere have been performed. In the present study, we compared the rhizosphere survival of the denitrifying Pseudomonas fluorescens YT101 strain with that of its isogenic mutant deficient in the ability to synthesize the respiratory nitrate reductase, coinoculated in nonplanted or planted soil. We demonstrated that under nonlimiting nitrate conditions, the denitrifying wild-type strain had an advantage in the ability to colonize the rhizosphere of maize. Investigations of the effect of the inoculum characteristics (density of the total inoculum and relative proportions of mutant and wild-type strains) on the outcome of the selection demonstrated that the selective effect of the plant was expressed only during the phase of bacterial multiplication and that the intensity of selection was dependent on the magnitude of this phase. Moreover, application of the de Wit replacement series technique to our results suggests that the advantage of the wild-type strain was maximal when the ratio between the two strains in the inoculum was close to 1:1. This work constitutes the first direct demonstration that the presence of a functional structural gene encoding the respiratory nitrate reductase confers higher rhizosphere competence to a microorganism.

Relationships between root density of the African grass Hyparrhenia diplandra and nitrification at the decimetric scale: an inhibition-stimulation balance hypothesis.

Previous studies have shown that Lamto savannah exhibits two different types of nitrogen cycle with high and low nitrification sites and suggested that the perennial grass Hyparrhenia diplandra is responsible for this duality at a subpopulation level, with one ecotype being thought to be able to inhibit nitrification. The present work aimed to investigate the relationships between nitrification and the roots of H. diplandra at two scales. (i) Site-scale experiments gave new insight into the hypothesized control of nitrification by H. diplandra tussocks: the two ecotypes exhibited opposite influences, inhibition in a low nitrification site (A) and stimulation in a high nitrification site (B). (ii) Decimetric-scale experiments demonstrated close negative or positive relationships (in sites A or B, respectively) between the roots and nitrification (in the 0-10 cm soil layer), showing an unexpectedly high sensitivity of the nitrification process to root density. In both soils, the correlation between the roots and nitrification decreased with depth and practically disappeared in the 20-30 cm soil layer (where the nitrification potential was found to be very low). Therefore, the impact of H. diplandra on nitrification may be viewed as an inhibition-stimulation balance.

Disruption of narG, the gene encoding the catalytic subunit of respiratory nitrate reductase, also affects nitrite respiration in Pseudomonas fluorescens YT101.

The Pseudomonas fluorescens YT101 gene narG, which encodes the catalytic alpha subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar(-) mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N(2)O respiration was not affected, anaerobic growth with NO(2) as the sole electron acceptor was delayed for all of the Nar(-) mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.

Purification of the dissimilative nitrate reductase of Pseudomonas fluorescens and the cloning and sequencing of its corresponding genes.

The dissimilative membrane-bound nitrate reductase from Pseudomonas fluorescens strain AK15 was purified and the alpha subunit of the enzyme partially sequenced. On the basis of this partial amino acid sequence and of conserved stretches of amino acids between Escherichia coli and Bacillus subtilis, degenerate primers were design to amplify the narG gene and part of the narH gene in a PCR approach. The deduced amino acid sequence of narG shows 72% and 52% and narH 78% and 62% identity to the homologous subunit of E. coli and B. subtilis, respectively.

Influence of Two Plant Species (Flax and Tomato) on the Distribution of Nitrogen Dissimilative Abilities within Fluorescent Pseudomonas spp.

The distribution of nitrogen-dissimilative abilities among 317 isolates of fluorescent pseudomonads was studied. These strains were isolated from an uncultivated soil and from the rhizosphere, rhizoplane, and root tissue of two plant species (flax and tomato) cultivated on this same soil. The isolates were distributed into two species, Pseudomonas fluorescens (45.1%) and Pseudomonas putida (40.4%), plus an intermediate type (14.5%). P. fluorescens was the species with the greatest proportion of isolates in the root compartments and the greatest proportion of dissimilatory and denitrifying strains. According to their ability to dissimilate nitrogen, the isolates have been distributed into nondissimilatory and dissimilatory strains, nitrate reducers and true denitrifiers with or without N(inf2)O reductase. The proportion of dissimilatory isolates was significantly enhanced in the compartments affected by flax and tomato roots (55% in uncultivated soil and 90 and 82% in the root tissue of flax and tomato, respectively). Among these strains, the proportion of denitrifiers gradually and significantly increased in the root vicinity of tomato (44, 68, 75, and 94% in uncultivated soil, rhizosphere, rhizoplane, and root tissue, respectively) and was higher in the flax rhizoplane (66%) than in the uncultivated soil. A higher proportion of N(inf2)O reducers was also found in the root compartments. This result was particularly clear for tomato. It is hypothesized that denitrification could be a selective advantage for the denitrifiers in the root environment and that this process could contribute to modify the specific composition of the bacterial communities in the rhizosphere.

Use of tn5 mutants to assess the role of the dissimilatory nitrite reductase in the competitive abilities of two pseudomonas strains in soil.

We examined the influence of soil aeration state and plant root presence on the comparative survival of wild-type bacteria and isogenic Tn5 (Nir(sup-)) mutants lacking the ability to synthesize nitrite reductase. Two denitrifying Pseudomonas strains with different nitrite reductase types were used. Enumeration of bacteria in sterile and nonsterile soils was based on differential antibiotic resistance. The validity of the bacterial models studied (i.e., equal growth of wild-type and mutant bacteria under aerobic conditions and significantly better growth of wild-type bacteria under denitrifying conditions) was verified in pure-culture studies. In sterile soil, both strains survived better under aerobic than under anaerobic conditions. The lower efficiency of denitrification than O(inf2) respiration in supporting bacterial growth explained this result, and the physical heterogeneity of soil did not strongly modify the results obtained in pure-culture studies. In nonsterile soil, one of the Pseudomonas strains survived better under anaerobic conditions while the other competed equally with the indigenous soil microflora under aerobic and anaerobic conditions. However, when the Nir(sup-)-to-total inoculant ratios (wild type plus Nir(sup-) mutant) were analyzed, it appeared that the presence of nitrite reductase conferred on both Pseudomonas strains a competitive advantage for anaerobic environment or rhizosphere colonization. This is the first attempt to demonstrate with isogenic nondenitrifying mutants that denitrification can contribute to the persistence and distribution of bacteria in fluctuating soil environments.

Delayed NH3 and N2 O uptake by maize leaves.

15 N was used to give evidence of NH3 and N2 O absorption by maize leaves. A short-term labelling method (90 min) was chosen with high NH3 concentration (40 µ1 1-1 ) to assess uptake mechanisms. Two initial entry pathways were suggested for NH3 : fast uptake with immediate metabolism and fast storage with progressive metabolism. A storage compartment delayed NH3 absorption after exposure. Similar mechanisms might be involved in N2 O uptake.