Bioremediation is a key technology for the attenuation of industrial scale xenobiotic production. Central to the optimisation of biological detoxification strategies is the monitoring of the efficiency of remediation and the impact of pollutant loading upon the key micro-organisms within the remediating consortia (Whiteley and Bailey, 2000). While chemical determinations provide an efficient means to measure pollutant attenuation, they only provide circumstantial evidence of the functional status of the degradative microbial communities. An alternative strategy for monitoring process efficacy and the activity of the remediating community is the selection of indicator organisms through ecological analyses of the system. In this study, we report on an ecological analysis to facilitate the selection of culturable isolates that have potential for further development of biosensors modified to carry reporter genes such as those for bacterial bioluminescence.

The degradation of phenolic wastes from industrial processes has been described in some detail for the optimisation of industrial bioremediation systems (Moreno and Sutton) to prevent the environmental damage caused by the deliberate or accidental discharge of industrial waste (Yang et al., 1998). One key component of microbial communities responsible for degradation of phenolic wastes are Pseudomonad species. The physiological and genetic basis of phenol degradation has been described for a number of Pseudomonas spp. (e.g. Kotturi; Nurk; Topp; Kiyohara; Motzkus; Arquiaga; Puhakka; Srivastava; Buitron and Loeser). The ubiquity and functionality of pseudomonads make them ideal candidates as process indicator organisms and to produce ecologically relevant biosensors.

Biosensors fall into two categories, specific and generalised. Specific sensors have been developed for the detection of one or more related compounds by utilising reporter gene constructs expressed from inducible promoters (e.g. Belkin and Corbisier). Generalised sensors have more widespread utility since they monitor the impact of toxicity on the overall physiological state of the cell, where a decrease in physiology correlates to an increase in toxicity (Paton and Boyd). Currently generalised sensors, although able to measure gross changes in remediation efficiency, are non-specific and based upon a particular isolate, which tend not to be native to the system under study, such as the application of marine bioluminescent bacteria (Kafka and Britz) or are required to respond to a diversity of mixed pollutants in a wide variety of environmental samples (Stom et al., 1992). Clearly, the use of ecological analyses to determine specific strains from natural process communities for potential biosensor design is an advantageous and beneficial approach in terms of sensor specificity and performance.

In our own investigations we concentrated on the analysis of the microbial community structure of an industrial plant that remediates phenolics (Whiteley and Bailey, 2000). To better understand the processes involved and to potentially develop appropriate toxicity assays to monitor discharge we have evaluated the physiological and community structuring of the Pseudomonad isolates, the dominant culturable component within the specialised degradative community, through rapid analysis of physiological response of a large number of isolates to primary pollutants.

2. MATERIALS AND METHODS

2.1. Sampling collection and strain identification

Samples were taken from an undisclosed industrial Vitox biological reactor in the UK as previously documented (Whiteley and Bailey, 2000). Typical operating input levels of phenolic species to the reactor, as determined by gas chromatography were between 250 and 500 mg/l.

Two parallel reactors were sampled by removal of reactor liquor and suspended solids. The solids (flocs) were separated from the liquor by centrifugation and bacteria suspended from each component by vortex disruption with glass beads (1 g of 5-mm beads in 10 ml of reactor liquor and 10 ml of phosphate buffer). Following mechanical agitation, 100 l of a decimal serial dilution of cell suspensions were plated to Pseudomonad selection agar (PSA) or tryptic soy broth (TSBA) (Difco, Oxoid, UK) both solidified with 1.5% (w/v) agar. Each agar (PSA or TSBA) was also supplemented with phenol (300 mg/l) or with 0.2 m filtered reactor liquor in order to provide a wider range of relevant isolation conditions. All manipulation and plating was carried out on site immediately after sampling to minimise artifacts introduced during storage or transportation. Plated samples were incubated at 28°C and colonies patched to TSBA plates after 48 h. Strain purity was ensured by re-plating colonies on fresh TSBA prior to storage under glycerol at −70°C. All analyses were conducted on fresh cultures derived from glycerol stocks to avoid physiological or biochemical changes that can occur by sub-culturing in the laboratory.

2.2. Identification of isolates by FAME-MIS

Isolates were identified by fatty acid methyl-ester (FAME) gas chromatography (GC) analysis using Microbial Identification Systems Software (MIS, Delaware, USA). Isolates were grown on TSBA for 24 h at 28°C and 50 mg wet weight of cells harvested for fatty acid extraction and analysis as described previously (Thompson et al., 1993).

2.3. Respiration assay of isolates in the presence of phenol

A single fresh colony picked from a TSBA plate incubated overnight at 28°C was inoculated into 5 ml of Luria Bertani broth and grown at 28°C with shaking at 220 r.p.m. In mid-exponential growth, OD_{610 nm}=0.5, phenol was added to final concentrations of 0, 50, 100, 200 or 400 mg/l and further incubated for 1 h at 28°C. MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma, St. Louis, MO, USA) was added to a concentration of 1.5 mM (Sigma, Dorset) to measure respiratory activity. The cellular respiration rates of the cultures in the presence of phenol, as a function of MTT reduction, were measured in a Bioscreen-C kinetic plate reader (Labsystems International, UK) by dual wavelength absorbance measurements at 492 and 610 nm over a 6-h period under constant shaking. Parallel samples from the same cultures without the addition of MTT were also measured over the 6-h period for absorbance at 610 nm to compare rates of cell division. MTT reduction rates were calculated as the increase in absorbance during incubation and expressed as a percentage of the toxicant free control value. This allowed normalisation and direct comparison of the activity response of the diverse isolates in the collection.

2.4. Utilisation of phenol as a sole carbon source

To test the ability of isolates to utilise phenol as a sole carbon source, cultures were inoculated onto a minimal salts medium (M9) (Maniatis et al., 1982) supplemented with 50–600 mg/l phenol and solidified with 1.5% agar. A range of concentrations of phenol were employed in order to encompass a balance between utilisation and toxicity of phenol for the range of strains assayed. Controls included the same cultures grown on M9 media supplemented with glucose (15 g/l).

2.5. Assessment of catechol 2,3 dioxygenase activity

The ability of the isolates to reduce a 1% (w/v) aqueous solution of Catechol (Sigma, UK) was used as a secondary measure to test for the presence of catechol 2,3 dioxygenase, an enzyme of the phenol degradative pathway. Colonies were sampled by filter lift from plates and sprayed with catechol. The appearance of a yellow-brown pigment within 10-min incubation at room temperature inferred catechol 2,3 dioxygenase activity.

2.6. Strain characterisation by REP-PCR

The clonal diversity of each identified species group was assessed by REP-PCR under standard conditions. DNA was recovered from each isolate (Bailey, 1995) and 50 ng amplified using the REP1R-1 and REP2-I primer pair, under standard conditions (Louws et al., 1994), in a final volume of 25 l employing a PTC225 thermal cycler (MJ Research Instruments, Watertown, MA). Amplicon fragments were separated by electrophoresis in 1% (w/v) agarose gels. After staining in ethidium bromide, gel, images were captured (BioRad 1000 GelDoc system, BIO-RAD, Hercules, CA) and band profiles analysed using the Phoretix 1D analysis software according to the manufacturers instructions (Phoretix International, Newcastle upon Tyne, UK). Dendrograms for comparisons between REP-PCR profiles were generated by using peak position matching using the Dice coefficient and trees generated by the UPGMA algorithm within the Phoretix software.

3. RESULTS

3.1. Isolation and identification of Pseudomonas spp. in bioreactor samples

The 700 bacteria isolated from the biological treatment plant under investigation were identified by FAME-GC analysis and represented 59 species groups. Pseudomonads, the dominant fraction, were classified into five Pseudomonas species groups and one newly re-classified Pseudomonad group, Brevundimonas vesicularis (Fig. 1). The P. pseudoalcaligenes and P. vesicularis groups accounted for the most abundant species isolated (16 and 15 strains, respectively, Table 1). Further, the distribution of the P. pseudoalcaligenes and P. vesicularis groups within the reactor were mostly confined to the suspended solid floc component of the ecosystem, where as P. syringae were only found in the supernatant liquor (Table 1).

 

Fig. 1. UPGMA dendrogram of 48 of the Pseudomonad isolates, classified by FAME analyses, from the total microbial diversity of the 700 mixed taxa isolates from the industrial bioreactor. Pseudomonad isolates highlighted in bold utilise phenol as a sole source of carbon and reduce catechol to a yellow semi-aldehyde. Note that P. vesicularis has been re-classified as Brevundimonas vesicularis.

 

Table 1. Characterisation of the main species groups of cultured pseudomonads by FAME analyses (Fig. 1), by isolation frequency from the suspended solids (floc) or rector liquor (supernatant) in relation to their ability to degrade and utilise phenol as a sole carbon source
 

 

3.2. Utilisation of phenol as a sole carbon source

Only those isolates able to utilise phenol as a sole carbon source on M9 media also had catechol dioxygenase activity. Following the analysis of the 48 selected isolates the ability to utilise phenol was mostly associated with the P. pseudoalcaligenes cluster (Table 1).

3.3. Respiration assay of isolates in the presence of phenol

To determine the susceptibility of bacteria to phenolic loading a rapid respiratory response assay was performed on 48 isolates representative of each Pseudomonas species group. Data are presented as the summed average for all isolates analysed from each grouping. The P. stutzeri group isolates were the most sensitive to increasing phenolic load as determined by the rapid rate of decline for MTT reduction above phenol concentrations of 50mg/l (Fig. 2). Conversely, the P. fluorescens and P. putida isolates were effectively insensitive to phenol and showed virtually no decrease in the respiratory response over the concentration range examined, up to 400 mg/l. This is equivalent to over three times the normal operating concentration of phenolics in the bioreactor (Whiteley and Bailey, 2000). Intermediate respiratory tolerances were observed for the remaining three species group, P. pseudoalcaligenes, P. vesicularis and P. syringae, all exhibited similar profiles of respiratory inhibition with increasing phenolic loading in comparison to control values (Fig. 2). Data for the analyses of individuals within each species group were performed after sub-division into those that were tolerant of and able to degrade phenol based on the EC₅₀ value obtained (EC₅₀ was determined as the phenol concentration (mg/l) required to reduce the respiration rate of the isolate by 50%). The phenol utilising isolates were mostly assigned to the P. pseudoalcaligenes (14 degraders from 16 isolates assayed (Table 1)). Within this group, the response of cell respiration to phenol loading of the non-degraders was not significantly different for those members which could utilise phenol (EC₅₀ values of 381mg/l, c.f. 294 mg/l, Table 2). By contrast those isolates from the P. fluorescens and P. putida group, that were able to utilize phenol, were also more resistant to phenol toxicity than the non-utilising members of the group. These differences were approximately two-fold higher when EC₅₀ values were compared (Table 2). The remaining species group, P. vesicularis comprised mainly non-degrading species where the sensitivity to phenol toxicity was not significantly different to the single isolate able to utilize phenol as a sole carbon source. Isolates of P stutzeri and P. syringae did not possess the phenol-degrading enzyme, but showed considerable differences in their range of respiratory tolerance.

 

 

Fig. 2. Respiration assays, MTT reduction as an indicator of respiration sensitivity to phenol loading. Plots expressed as a percentage of control values (no toxicant) and averaged for all isolates within a species group (minimum n=3 isolates, maximum n=16 isolates). Note: The Y-axis for P. fluorescens response has been extended to cover the range of response for this group, a smaller range for the respiration rate is presented to better illustrate response to increasing phenol concentrations.

 

Table 2. Summary of respiration assays expressed as an EC₅₀ value for isolates identified as phenol degraders (utilisation of phenol as a sole source of carbon) and non-degraders

The EC₅₀ values presented indicate the quantity of phenol (mg/l) required to reduce the respiration of the isolates to 50% of control (no toxicant) values. Values indicating >400 mg/l are for isolates where no visible effect on respiration was observed after incubation in the presence of up to 400 mg/l phenol.

–=no degradation observed.
 

 

3.4. Strain characterisation by REP-PCR

The genotype of isolates was determined to assess the extent of clonality in the sampled populations. Considerable genotypic variation was observed within and between species groups established by phenotypic assessment of the FAME content of isolates. The comparison of REP-profiles was undertaken by pair-wise comparison of the fragment patterns and the relationship between the isolates determined (Fig. 3). For example, the principal degrader isolates within the P. seudoalacaligenes group varied by up to 50% in genotype pattern, indicative of variation in their genome organisation (Fig 3). However, within this group some isolates clustered at the 70% level and above. No correlation could be drawn between genotype and distribution within either the liquor or suspended solids (flocs). In general, the phenotypic data supported strong species grouping whereas genotypic comparison indicated genetic diversity typical of a complex community of functionally active strain variants.

 

Fig. 3. Genotypic relationship between pseudomonad isolates based upon REP-PCR profiles. The dendrogram was generated by pair-wise comparison of peak presence and position using the DICE coefficient and UPGMA algorithm, those isolates able to utilise phenol as a sole source of carbon are highlighted in bold.

 

4. DISCUSSION

We have presented a detailed analyses of the genetic and phenotypic diversity of Pseudomonad species isolated from a highly active phenolic degrading environment and assessed their ability to degrade phenol, the major pollutant in the system. The diversity observed supports the presumption of the complexity of the system and underlines the extent of phenotypic and genotypic plasticity of Pseudomonas spp. as has been reported in a number of highly selective environments. Although some criticism is made of the isolation approach, it does provide an effective comparative measure. While it is accepted that culture dependant methods introduce bias (Wagner; Dunbar and Watanabe) it is important to stress that such approaches are necessary as the evaluation of biochemical and physiological parameters allows the characterisation of isolates with relation to both treatment efficacy and community structure/function (Schleifer; Buitron; Watanabe; Duteau and Watanabe).

A key observation made was the apparent niche specialisation for some isolates within the community. The P. pseudoalcaligenes comprise the majority phenol utilising isolates although there was no observable relationship between the ability to degrade phenol and the respiratory tolerance of the organisms to phenolic loading, indicating that the majority of the degraders were relatively sensitive to phenol loading. These assessments were typical of the tolerance range observed previously in Pseudomonads by culture methods (Kapoor et al., 1998). Indeed, the majority of degraders (P. pseudoalcaligenes) actually had a lower tolerance to phenolic loading than other species groups, which could not degrade phenol. Ecologically this niche separation (in terms of functional compartmentalisation, physiological tolerance and degradative ability) constitutes an important driver of community structure and functionality. For example, an increasing in phenolic load in the system under study could reduce efficiency in the degradative population due to direct toxicity effects in the susceptible P. pseudoalcaligenes group. Possible consequences of an increase in the main pollutant would be the selection for a less diverse degradative community or the selection of more tolerant groups that were non-degrading, which in turn would result in plant failure as concentrations of phenolics became toxic to all the bacteria. Whether such diversity changes would in fact directly impact on community functionality (e.g., Watanabe et al., 1999) or be compensated for by further diversity re-assortment (Fernandez et al., 1999) remains unresolved and requires further rigorous testing.

Analyses of the genetic background of the phenol utilising isolates was undertaken to assess diversity. REP-PCR fingerprint analysis demonstrated divergence in genome organisation, suggesting either a relatively imprecise resolution during the initial strain characterisation by FAME, or, more likely the niche selection of sub-species. This sub-division may also reflect the genotypic and phenotypic plasticity of each group, particularly as the ability to degrade phenol has frequently been associated with lateral gene transfer (Nurk; Powlowski; Herrmann; Kallastu and Yoon).

The analyses of isolates from the bioreactor revealed several important traits related to the diversity, physiology and function of the pseudomonad community present in the phenol-degrading industrial bioreactor under study. Considerable physiological heterogeneity was observed in the tolerance range of the isolates to the major pollutant. This variety provides isolates with a range of sensitivity that may be effective in the design of specific biosensors able to report directly on the defined process under study. The rapid physiological screening assays described here provide a rapid approach for the selection of candidate isolates for modification. Modification includes the insertion of reporter genes, such as those for bioluminescence, to provide a suite of biosensors with varying levels of tolerance to the pollutant. Respiration analyses indicated that organisms belonging to the P. stutzeri species group could serve as key indicators of remediation efficacy and consortia physiological status since their physiology is the most sensitive to phenolic loading. In contrast, organisms belonging to the P. fluorescens or P. putida groupings may serve as indicators of extreme toxicity. Biosensors derived from the P. pseudoalcaligenes, P. vesicularis and P. syringae, through their mid-range physiological tolerances constitute candidates with a capability to predict initial phases of extreme toxicity or reinforce the latter phases of gradual toxicity increases detected by the lower range P. stutzeri based sensors.

ACKNOWLEDGEMENTS

This work was supported by the Natural Environment Research Council and the LINK-BTSW (Biological Treatment of Soil and Water) initiative. The authors wish to thank Malcolm Fisher and Paul Whitby for sample access and discussions regarding the bioreactor.

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