Inhibition of biological phosphorus removal in a sequencing moving bed biofilm reactor in seawater.

A new process was developed to achieve denitrifying biological phosphorus removal in wastewaters containing high levels of nitrate and phosphate with a low level of organic matter. This could particularly be useful in recirculating systems such as aquariums or fish farms to prevent accumulation of nitrate and phosphates and to avoid regular cost extensive and polluting water replacement. Phosphorus (P) was removed from the influent in a sequencing moving bed biofilm reactor, stored in the attached biomass and then cyclically removed from the biomass by filling the reactor with anaerobic water from a stock tank. Phosphate was accumulated in the stock tank which allowed for use as fertilizer. The feasibility of the experimental design was demonstrated by using the activated sludge model No. 3 (ASM3) complemented by the EAWAG Bio-P module implemented in the WEST simulation software. A pilot scale experiment was conducted in two identical reactors in two runs: one to treat water from a marine mesocosm, the other to treat a synthetic freshwater influent. No biological phosphorus removal was achieved during the seawater run. During the freshwater run, average P removal efficiency was 20%, of which 80% was attributed to biological removal and 20% to chemical precipitation. The absence of efficiency in seawater was attributed to the high concentration of calcium.

Molecular analysis of Desulfitobacterium frappieri pcp-1 involved in reductive dehalogenation of pentachlorophenol.

Desulfitobacterium are Gram positive, spore-forming, strictly anaerobic bacteria, that belong to the Firmicutes, Clostridia, Clostridiales, and Peptococcaceae. Most known members of the genus Desulfitobacterium have the ability to dechlorinate several halogenated compounds by a mechanism of reductive dehalogenation and use them as electron acceptors to generate energy (halorespiration). Desulfitobacteria are therefore perfect candidates to be used in bioremediation treatments of environment polluted with halogenated compounds. Understanding the physiology and the molecular mechanisms of these bacteria will help to develop better bioremediation systems. This report summarizes works that have been done in our laboratories with D. frappieri PCP-1 on reductive dehalogenases, genes encoding these dehalogenases and their expression, and the development of lab-scale PCP-degrading reactors using this bacterium.

Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor.

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.

Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste.

The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques. The temperature increased quickly in the first hours of the treatment cycles and values up to 72 degrees C were reached. Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles. A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60 degrees C. Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found. In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%). The other Bacilli could not be assigned to a known species. Schineria larvae was the second most abundant species (14%) in the clone library. Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40 degrees C and 60 degrees C. Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated.

Bacterial diversity in a marine methanol-fed denitrification reactor at the montreal biodome, Canada.

The bacterial biota of a methanol-fed denitrification reactor used to treat seawater at the Montreal Biodome were investigated using culture-dependent and molecular biology methods. The microbiota extracted from the reactor carriers were cultivated on three media. Three isolate types were recovered and their 16S ribosomal DNA (rDNA) genes were determined. The analysis showed that the isolate types were related to alpha-Proteobacteria. They are members of the Hyphomicrobium and Paracoccus genera and the Phyllobacteriaceae family. Uncultured bacteria were identified through a 16S rDNA library generated from total DNA extracted from the microbiota. Clones were screened for different restriction profiles and for different DGGE (denaturing gradient gel electrophoresis) migration profiles. More than 70% of clones have the same restriction profile, and the sequence of representative clones showed a relation with the Methylophaga members of the Piscirickettsia family (gamma-Proteobacteria). Sequences from other profiles were related to bacterial species involved in denitrification. The number of species in the denitrification reactor was estimated at 15. Bacterial colonization on newly added carriers in the denitrification reactor was monitored by PCR-DGGE. The DGGE migration profiles evolved during the first 5 weeks and then remained essentially unchanged. PCR-DGGE was also used to monitor the microbial profiles in various aquarium locations. As expected, bacterial populations differed from one location to another, except for the sand and trickling filters which presented similar DGGE migration profiles.

Initial characterization of new bacteria degrading high-molecular weight polycyclic aromatic hydrocarbons isolated from a 2-year enrichment in a two-liquid-phase culture system.

AIMS: To characterize some polycyclic aromatic hydrocarbons (PAH)-degrading microorganisms isolated from an enriched consortium degrading high molecular weight (HMW) PAHs in a two-liquid-phase (TLP) soil slurry bioreactor, and to determine the effect of low molecular weight (LMW) PAH on their growth and HMW PAH-degrading activity. METHODS AND RESULTS: Several microorganisms were isolated from a HMW-PAH (pyrene, chrysene, benzo[a]pyrene and perylene) degrading consortium enriched in TLP cultures using silicone oil as the organic phase. From 16S rRNA analysis, four isolates were identified as Mycobacterium gilvum B1 (99% identity),Bacillus pumilus B44 (99% identity), Microbacterium esteraromaticum B21 (98% identity), and to the genus Porphyrobacter B51 (96% identity). The two latter isolates have not previously been associated with PAH degradation. Isolate B51 grew strongly in the interfacial fraction in the presence of naphthalene vapours and phenanthrene compared with cultures without LMW PAHs. Benzo[a]pyrene was degraded in cultures containing a HMW PAH mixture but pyrene had no effect on its degradation. The growth of isolates B1 and B21 was improved in the aqueous phase than in the interfacial fraction for cultures with naphthalene vapours. Pyrene was required for benzo[a]pyrene degradation by isolate B1. For isolate B21, pyrene and chrysene were degraded only in cultures without naphthalene vapours. CONCLUSION: Consortium enriched in a TLP culture is composed of microorganisms with different abilities to grow at the interface or in the aqueous phase according to the culture conditions and the PAH that are present. Naphthalene vapours increased the growth of the microorganisms in TLP cultures but did not stimulate the HMW PAH degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: New HMW PAH-degrading microorganisms and a better understanding of the mechanisms involved in HMW PAH degradation in TLP cultures.

Strategies for augmenting the pentachlorophenol degradation potential of UASB anaerobic granules.

Anaerobic degradation of pentachlorophenol (PCP) is an example of a process that may benefit from enrichment or bioaugmentation. In one approach, enrichment acceleration was attempted by applying an on-line control-based selective stress strategy to a native anaerobic upflow sludge bed (UASB) system; this strategy linked PCP loading rate to methane production. As a result, the reactor biomass potential for PCP complete dechlorination reached a rate of 4 mg g(-1) volatile suspended solid (VSS) day(-1) within a period of 120 days. In another approach, a pure culture, Desulfitobacterium frappieri PCP-1, a strictly anaerobic Gram-positive bacterium, was used to augment the granular biomass of the UASB reactor. This also resulted in a specific degradation rate of 4 mg PCPg(-1) VSS day(-1); however, this potential was attained within 56 days. Fluorescent in situ hybridization (FISH) showed that the PCP-1 strain was able to rapidly attach to the granule and densely colonize the outer biofilm layer.

Microstructure of anaerobic granules bioaugmented with Desulfitobacterium frappieri PCP-1.

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day(-1) and a low bacterial diffusion of 10(-7) dm(2) day(-1). Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.

Geographic distribution of Desulfitobacterium frappieri PCP-1 and Desulfitobacterium spp. in soils from the province of Quebec, Canada.

The presence of indigenous Desulfitobacterium species in 44 soil samples taken from various sites in the southern part of the province of Quebec (Canada) and four from locations outside Quebec was investigated. Twenty-four of these soils were sampled from contaminated industrial sites. Indigenous Desulfitobacterium bacteria from soil samples were enriched by cultivation in anaerobic soil slurry culture. Total DNA was then extracted from these slurries and polymerase chain reaction (PCR) amplifications were performed with primers targeting 16S ribosomal RNA gene sequences of Desulfitobacterium spp. and of Desulfitobacterium frappieri PCP-1. A positive PCR signal was obtained in 31 soil slurry cultures. Resolution of single-strand DNAs of some of the PCR products by a single-strand conformational polymorphism protocol suggests that more than one species of Desulfitobacterium were present in the corresponding slurry cultures. These results suggest that Desulfitobacterium are ubiquitous in soils in the province of Quebec, especially in soils from the St. Lawrence valley and the southern part of the province.

Separation of a phenol carboxylating organism from a two-member, strict anaerobic co-culture.

In a culture converting phenol to benzoic acid under anaerobic conditions and previously described as being constituted of only a Clostridium-like strain 6, another bacterium (strain 7) was observed. Each organism was enriched by centrifugation on a Percoll gradient. Strain 6 was purified by dilution and plating. Strain 7 did not grow on solid media, but a strain 7 culture, cleared of strain 6, was obtained by subculturing in the presence of ampicillin and by dilution. In fresh medium, phenol was transformed by the reconstituted co-culture but not by each strain alone. In a supernatant from a co-culture or from a strain 6 culture, strain 7 alone transformed phenol but not strain 6. Maintenance of an active strain 7 in fresh medium instead of co-culture supernatant became possible when phenol was replaced by 4-hydroxybenzoate (4-OHB), which is decarboxylated to phenol before being transformed to benzoate. Even with 4-OHB, the use of co-culture (or strain 6 culture) supernatant resulted in faster transformation activity and growth rate. A phylogenetic analysis placed strain 7 in a cluster of uncultivated or nonisolated bacteria (92-96% homology). Strain 7 is also related to Desulfotomaculum, Desulfitobacterium, Desulfosporosinus, Moorella, and Sporotomaculum genera (87-92% homology).

Hydrolysis of 4-hydroxybenzoic acid esters (parabens) and their aerobic transformation into phenol by the resistant Enterobacter cloacae strain EM.

Enterobacter cloacae strain EM was isolated from a commercial dietary mineral supplement stabilized by a mixture of methylparaben and propylparaben. It harbored a high-molecular-weight plasmid and was resistant to high concentrations of parabens. Strain EM was able to grow in liquid media containing similar amounts of parabens as found in the mineral supplement (1,700 and 180 mg of methyl and propylparaben, respectively, per liter or 11.2 and 1.0 mM) and in very high concentrations of methylparaben (3,000 mg liter(-1), or 19.7 mM). This strain was able to hydrolyze approximately 500 mg of methyl-, ethyl-, or propylparaben liter(-1) (3 mM) in less than 2 h in liquid culture, and the supernatant of a sonicated culture, after a 30-fold dilution, was able to hydrolyze 1,000 mg of methylparaben liter(-1) (6.6 mM) in 15 min. The first step of paraben degradation was the hydrolysis of the ester bond to produce 4-hydroxybenzoic acid, followed by a decarboxylation step to produce phenol under aerobic conditions. The transformation of 4-hydroxybenzoic acid into phenol was stoichiometric. The conversion of approximately 500 mg of parabens liter(-1) (3 mM) to phenol in liquid culture was completed within 5 h without significant hindrance to the growth of strain EM, while higher concentrations of parabens partially inhibited its growth.

Clogging of a limestone fracture by stimulating groundwater microbes.

Biological clogging is promoted in aquifers either to contain or to remediate groundwater. In this study, an apparatus able to detect small changes in hydraulic conductivity (K) was developed to measure the clogging of a single fracture in limestone, following microbial stimulation. The fracture had a 2.5 mm2 section and was 50 cm long. Prior to the inoculation of the limestone, the sequencing of representative clones from 16S rRNA genes isolated from groundwater, showed significant affiliation with Cytophaga spp., Arcobacter spp. and Rhizobium spp. These bacteria are known to secrete extracellular polymeric substances and form biofilms. When nutrients were added to the inoculated limestone, a decrease in K occurred after 8 days, reaching 0.8% of its initial value after 22 days (Kfi = 340 cm min-1). This study showed that a stimulation of indigenous microbes from groundwater effectively clogged a macrofracture in limestone, suggesting the potential application of biobarriers in fractured rock aquifers.

Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when P. aeruginosa 57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when P. aeruginosa 57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.

Assessment of Changes in Biodiversity when a Community of Ultramicrobacteria Isolated from Groundwater Is Stimulated to Form a Biofilm.

The stimulation of groundwater bacteria to form biofilms, for the remediation of polluted aquifers, is subjected to environmental regulations that include measurement of effects on microbial biodiversity. Groundwater microorganisms contain a proportion of unidentified and uncharacterized ultramicrobacteria (UMB) that might play a major role in the bioclogging of geological materials. This study aimed to assess the changes in genetic and metabolic biodiversity when a community of UMB, isolated from groundwater, is stimulated to form biofilms on a ceramic surface. UMB were stimulated with aerobic conditions and injection of molasses, in reactors reproducing groundwater composition and temperature. Concentration of planktonic viable UMB, secretion of extracellular polymeric substances (EPS), and biofilm thickness were monitored. The assessment of changes in biodiversity was achieved by comparing the initial UMB community to the biofilm community, using the single strand conformational polymorphism (SSCP) method, the cloning and sequencing of 16S rRNA gene (16S rDNA) sequences, and the Biolog microplate system. The hypothesis stating that indigenous UMB would play a significant role of in the biofilm development was corroborated. Within 13 days of stimulation, the UMB produced 700 mg L?1 of planktonic EPS and formed a biofilm up to a thickness of 1100 mm. This stimulation led to a decrease in genetic diversity and an increase in metabolic diversity. The decrease in genetic diversity was shown by a reduced number of single strand DNA fragments in the SSCP profiles. As such, 16S rDNA sequences from the biofilm revealed the predominance of four bacterial groups: Zoogloea, Bacillus/Paenibacillus, Enterobacteriaceae, and Pseudomonads. A significant increase in metabolic diversity was shown by a highest substrate richness profile and a lower substrate evenness profile of the biofilm bacterial population (p = 0.0 and p = 0.09, respectively). This higher metabolic diversity might be a consequence of the stimulation that seemed to favor the growth of bacteria having a high nutritional versatility. Stimulation of UMB, isolated from groundwater, was effective to form a biofilm having a high metabolic biodiversity. This combination of molecular-based and metabolic-based methods expanded the insight into monitoring the changes in bacterial biodiversity.

Two-liquid-phase slurry bioreactors to enhance the degradation of high-molecular-weight polycyclic aromatic hydrocarbons in soil.

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. The addition of a water-immiscible, nonbiodegradable, and biocompatible liquid, silicone oil, to a soil slurry was studied to promote the desorption of PAHs from soil and to increase their bioavailability. First, the transfer into silicone oil of phenanthrene, pyrene, chrysene, and benzo[a]pyrene added to a sterilized soil (sandy soil with 0.65% total volatile solids) was measured for 4 days in three two-liquid-phase (TLP) slurry systems each containing 30% (w/v) soil but different volumes of silicone oil (2.5%, 7.5%, and 15% [v/v]). Except for chrysene, a high percentage of these PAHs was transferred from soil to silicone oil in the TLP slurry system containing 15% silicone oil. Rapid PAH transfer occurred during the first 8 h, probably resulting from the extraction of nonsolubilized and of poorly sorbed PAHs. This was followed by a period in which a slower but constant transfer occurred, suggesting extraction of more tightly bound PAHs. Second, a HMW PAH-degrading consortium was enriched in a TLP slurry system with a microbial population isolated from a creosote-contaminated soil. This consortium was then added to three other TLP slurry systems each containing 30% (w/v) sterilized soil that had been artificially contaminated with pyrene, chrysene, and benzo[a]pyrene, but different volumes of silicone oil (10%, 20%, and 30% [v/v]). The resulting TLP slurry bioreactors were much more efficient than the control slurry bioreactor containing the same contaminated soil but no oil phase. In the TLP slurry bioreactor containing 30% silicone oil, the rate of pyrene degradation was 19 mg L(-)(1) day(-)(1) and no pyrene was detected after 4 days. The degradation rates of chrysene and benzo[a]pyrene in the 30% TLP slurry bioreactor were, respectively, 3.5 and 0.94 mg L(-)(1) day(-)(1). Low degradation of pyrene and no significant degradation of chrysene and benzo[a]pyrene occurred in the slurry bioreactor. This is the first report in which a TLP system was combined with a slurry system to improve the biodegradation of PAHs in soil.

Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture.

The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity from a phenol-carboxylating coculture, consisting of Clostridium-like strain 6 and an unidentified strain 7, was studied. Assays done with cell extracts showed that the optimal pH was 5.0-6.5 and the Km was 5.4 mM. The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and even enhanced by the addition of Mg++, Mn++, Zn++, or Ca++. After purification, the molecular mass of the enzyme was estimated as 420 kDa by gel chromatography, and as 119 kDa by SDS-PAGE, suggesting a homotetrameric structure. Its pI was 5.6. The N-terminal amino acid sequence showed 95% and 76% homology with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from Enterobacter agglomerans and Klebsiella pneumoniae, respectively. The purified enzyme also slowly catalyzed the reverse reaction, that is the phenol carboxylation. These characteristics suggest that this enzyme is different from other known decarboxylases. This includes the 4OHB-DC from Clostridium hydroxybenzoicum, which is the only one that had been purified before.

Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP.

Two rapid and simple methods for the characterisation and quantification of rhamnolipids produced by a growing culture of the Pseudomonas aeruginosa strain 57RP were developed. Two rhamnolipids were purified and their response factors determined. The various rhamnolipids produced were then measured using liquid chromatography/mass spectrometry. The culture supernatants were injected directly, without prior purification, in a HPLC equipped with a C(18) reverse-phase column. The complete profile of rhamnolipid congeners produced during a 2 week cultivation period was monitored. In order to shorten the analysis time, another method was developed which did not require chromatographic separation of the rhamnolipids prior to their detection. Quantification of rhamnolipids using the direct infusion method gave results very similar to those obtained with HPLC separation. These two methods were very well correlated with the standard colorimetric orcinol method. The rhamnolipid profiles obtained show that the various rhamnolipid congeners are secreted simultaneously, and that their relative proportion remained unchanged throughout the cultivation period.

Monitoring by laser-flow-cytometry of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. strain 107 during biotreatment of a contaminated soil.

A flow cytometric method (FCM) was used to detect and accurately enumerate a polycyclic aromatic hydrocarbon-degrading bacterial strain, Sphingomonas sp. 107, inoculated into a soil sample artificially contaminated with pyrene. To compare the FCM method with colony forming unit (CFU) assays, a rifampicin-resistant Sphingomonas sp. 107 was obtained which could be distinguished from the indigenous microflora, since there was no organism resistant to rifampicin in the soil that could transform indole to indigo (naphthalene dioxygenase activity). By combining light-scattering profiles (i.e., morphological properties), ethidium bromide influx (i.e., cell wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could enumerate the bacterial population of interest from the indigenous microflora and soil debris during the biotreatment. The FCM technique revealed that the number of inoculated Sphingomonas cells decreased gradually for 15 days of incubation before reaching a steady level of 7 to 12 x 10(5) cells.g-1 of soil. Similar values were obtained with the CFU assay. During this period, pyrene concentration decreased from 632 to 26 mg.kg-1 of dry soil. The FCM detection was improved by adding blocking reagent to the hybridization buffer to minimize the non-specific attachment of the fluorescent probe to soil particles. Combined with the improvements in probe technology, FCM detection was shown to be a good alternative to the conventional culture methods for the analysis of bacterial populations in environmental samples. This technique could be potentially useful for the detection of microorganisms that grow poorly in culture.

Optimization of high-molecular-weight polycyclic aromatic hydrocarbons' degradation in a two-liquid-phase bioreactor.

A microbial consortium degrading the high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) pyrene, chrysene, benzo[a]pyrene and perylene in a two-liquid-phase reactor was studied. The highest PAH-degrading activity was observed with silicone oil as the water-immiscible phase; 2,2,4,4,6,8, 8-heptamethylnonane, paraffin oil, hexadecane and corn oil were much less, or not efficient in improving PAH degradation by the consortium. Addition of surfactants (Triton X-100, Witconol SN70, Brij 35 and rhamnolipids) or Inipol EAP22 did not promote PAH biodegradation. Rhamnolipids had an inhibitory effect. Addition of salicylate, benzoate, 1-hydroxy-2-naphtoic acid or catechol did not increase the PAH-degrading activity of the consortium, but the addition of low-molecular-weight (LMW) PAHs such as naphthalene and phenanthrene did. In these conditions, the degradation rates were 27 mg l-1 d-1 for pyrene, 8.9 mg l-1 d-1 for chrysene, 1.8 mg l-1 d-1 for benzo[a]pyrene and 0.37 mg l-1 d-1 for perylene. Micro-organisms from the interface were slightly more effective in degrading PAHs than those from the aqueous phase.

Monitoring of Desulfitobacterium frappieri PCP-1 in pentachlorophenol-degrading anaerobic soil slurry reactors.

Anaerobic biodegradation of pentachlorophenol (PCP) was studied in rotative bioreactors containing 200 g of PCP-contaminated soil and 250 ml of liquid medium. Reactors were bioaugmented with cells of Desulfitobacterium frappieri strain PCP-1, a bacterium able to dehalogenate PCP to 3-chlorophenol. Cells of strain PCP-1 were detected by quantitative PCR for at least 21 days in reactors containing 500 mg of PCP per kg of soil but disappeared after 21 days in reactors with 750 mg of PCP per kg of soil. Generally, PCP was completely removed in less than 9 days in soils contaminated with 189 mg of PCP per kg of soil. Sorption of PCP to soil organic matter reduced its toxicity and enhanced the survival of strain PCP-1. In some non-inoculated reactors, the indigenous microorganisms of some soils were also able to degrade PCP. These results suggest that anaerobic dechlorination of PCP in soils by indigenous PCP-degrading bacteria, or after augmentation with D. frappieri PCP-1, should be possible in situ and ex situ when the conditions are favourable for the survival of the degrading microorganisms.