Experimental sulfate amendment alters peatland bacterial community structure.

As part of a long-term, peatland-scale sulfate addition experiment, the impact of varying sulfate deposition on bacterial community responses was assessed using 16S tag encoded pyrosequencing. In three separate areas of the peatland, sulfate manipulations included an eight year quadrupling of atmospheric sulfate deposition (experimental), a 3-year recovery to background deposition following 5years of elevated deposition (recovery), and a control area. Peat concentrations of methylmercury (MeHg), a bioaccumulative neurotoxin, were measured, the production of which is attributable to a growing list of microorganisms, including many sulfate-reducing Deltaproteobacteria. The total bacterial and Deltaproteobacterial community structures in the experimental treatment differed significantly from those in the control and recovery treatments that were either indistinguishable or very similar to one another. Notably, the relatively rapid return (within three years) of bacterial community structure in the recovery treatment to a state similar to the control, demonstrates significant resilience of the peatland bacterial community to changes in atmospheric sulfate deposition. Changes in MeHg accumulation between sulfate treatments correlated with changes in the Deltaproteobacterial community, suggesting that sulfate may affect MeHg production through changes in the community structure of this group.

PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies.

Phylogeny and organization of recombinase in trio (RIT) elements.

Recombinase in trio (RIT) elements are composed of three adjacent tyrosine based site-specific recombinases that commonly occur in bacterial genomes. In this study, we examine RIT elements found in the genomes of strains from 63 different genera across 7 phyla of Eubacteria and examine the specific organization of these elements, their phylogenetic and environmental distribution, and their potential for mobility. We have found that each recombinase in this RIT arrangement is associated with a distinct sub-family of the tyrosine recombinases, and that the order and orientation of these sub-families is consistently maintained. We have determined that the distribution of these elements suggests that they are an ancient feature of bacterial genomes, but identical copies found within individual strains indicates that they are capable of intragenomic mobility. The occurrence of identical elements on both the main chromosome and one or more plasmids within individual strains, coupled with the finding that in some cases related genera are carrying highly similar RIT elements indicates that horizontal transfer has in some cases proceeded through a plasmid intermediate.

The limitations of draft assemblies for understanding prokaryotic adaptation and evolution.

The de novo assembly of next generation sequencing data is a daunting task made more difficult by the presence of genomic repeats or transposable elements, resulting in an increasing number of genomes designated as completed draft assemblies. We created and assembled idealized sequence data sets for Cupriavidus metallidurans CH34, Caulobacter sp. K31, Gramella forsetii KT0803, Rhodobacter sphaeroides 2.4.1 and Bordetella bronchiseptica RB50. In addition to confirming the role of transposable elements in interrupting the assemblies, an association was found between the most fragmented regions and known or predicted genomic islands in these strains. Assembly quality was more strongly related to putative genomic island content than to any other factor examined. We believe this association indicates that draft assemblies are limiting our ability to understand the genomic context of important bacterial adaptations and that the increased effort required for finishing genomes can provide a wealth of information for future studies.

Role of high molecular mass organics in colour formation during biological treatment of pulp and paper wastewater.

This paper forms part of series of biological treatment colour behaviour studies. Surveys across a range of mills have observed colour increases in aerated stabilisation basins of 20-45%. Much of the colour formation has been demonstrated to occur in high molecular mass effluent organic constituents (HMM) present in bleach plant effluents. Removing material greater than 3000 Da essentially eliminated the colour forming ability in both E and D stage wastewaters. We have also shown that pulp and paper sludges contain anaerobic bacteria capable of reducing humic like materials. Colour formation was correlated to the anoxic conditions and the availability of readily biodegradable organic constituents during the wastewater treatment process. Overall, these studies suggest that colour formation in pulp and paper biological treatment systems may be caused by anaerobic bacteria using HMM material from the bleaching effluents as an electron acceptor for growth. This leads to the reduction of the material, which in turn leads to non-reversible internal changes, such as intra-molecular polymerisation or formation of chromophoric functional groups.

Effect of chemical and physical parameters on a pulp mill biotreatment bacterial community.

The bacterial community composition in an activated sludge plant treatment from a bleached kraft pulp mill was monitored over a period of 209 days. Using DGGE and terminal-Restriction Fragment Length Polymorphism (t-RFLP) analysis we generated community DNA fingerprints over the time period. Both methods produce fingerprints that can be used to monitor stability in the system and generate fragments that can be associated with bacterial taxa. Chemical and physical parameters were also collected during that same time frame. We found a number of significant correlations with influent variables such as temperature, chemical oxygen demand (COD), Biochemical oxygen demand (BOD) and chloroform concentrations suggesting that these were the most likely parameters to influence the bacterial community structure. In addition several taxa correlated to important performance indicators such as COD/BOD removals and SVI. Multivariate analysis also confirmed the strong links between taxa variation and temperature, nutrient loads, chloroform and also one class of filaments. Establishing the identity of these taxa and their ecological preferences will greatly enhance our understanding and management of biological treatment systems.

The effects of cyanobacterial exudates on bacterial growth and biodegradation of organic contaminants.

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.

The impacts of cyanobacteria on pulp-and-paper wastewater toxicity and biodegradation of wastewater contaminants.

This study investigated the effects of cyanobacteria from pulp-and-paper waste-treatment systems on biological toxicity removal and biodegradation of certain wastewater contaminants. In field and batch studies, using the Microtox assay, cyanobacterial biomass and final wastewater toxicity were significantly correlated. In softwood-based wastewater, a decrease in toxicity was negatively correlated with cyanobacterial biomass, but the correlation was positive in hardwood-based wastewater. In the softwood-based wastewater, toxicity remained higher in the light than it was in the dark, whereas in hardwood-based wastewater, toxicity was lower in the light than it was in the dark. All of these results were light-dependent, suggesting that the photosynthetic growth of cyanobacteria is required to induce significant effects. When grown in mixed cultures with bacterial degraders, cyanobacteria from pulp-and-paper waste-treatment systems generally impeded the biodegradation of the wastewater contaminants phenol and dichloroacetate (DCA). However, there was one case where the cyanobacterium Phormidium insigne improved the bacterial degradation of DCA. Doubling inorganic nutrient concentrations did not improve phenol or DCA biodegradation in the majority of cases, indicating that nutrient competition is not a major factor. These data suggest that cyanobacteria play an important role during the biological treatment of contaminants, and, hence, toxicity removal in pulp-and-paper waste-treatment systems.

The formation of colour during biological treatment of pulp and paper wastewater.

Colour discharges are gaining renewed focus in the pulp and paper industry as increasingly strict regulatory limits are placed on wastewater quality and aesthetics. In-mill process improvements, such as ECF bleaching and oxygen delignification, have decreased wastewater colour loadings. However, a survey of 12 pulp and paper mill systems found that effluent treatment using aerated stabilisation basins (ASB) leads to average increases in colour of 20-40%. In some instances, this phenomenon may even double the influent colour levels. Activated sludge systems did not produce a colour increase. The measured increases that follow ASB secondary treatment may be sufficient for a mill to fail prescribed discharge standards. A detailed field survey focusing on sections of an integrated bleached kraft mill ASB treatment system was undertaken. The average increase in colour at the final point of discharge was 45%. The major changes in colour concentration occurred in the inlet to the main treatment pond, and in polishing ponds that followed the main treatment pond. Both of these areas receive little or no aeration. No significant change was observed in the highly aerated main pond. These results, along with literature reports, suggested that redox conditions play a major role in influencing colour behaviour. To test this, two series of paired continuously stirred reactors were used to treat whole mill effluent from two ECF bleached kraft mills in parallel. The first series initially treated under anaerobic conditions, followed by an aerobic reactor, while the second series reversed this order. With the initial anaerobic stage, effluent colour increased by 18% and 19% for the first and second series respectively. Subsequent treatment by aerobic bacteria further increased colour by 14% and 6%, for a total increase of 32% and 25%. Initial aerobic treatment, however, did not lead to any significant change in colour for either effluent. Further anaerobic treatment following aerobic conditions produced only small increases in colour. These results are consistent with the ASB and activated sludge system survey, suggesting that anaerobic conditions at the head of treatment systems initiate the observed increases in effluent colour in ASB treatment systems.

Treatment of volatile organic compounds in a biotrickling filter under thermophilic conditions.

The objectives of this research were to investigate the potential to biologically treat volatile organic compounds emitted by the forest products industry at thermophilic conditions and to examine the microbial community developed at high temperatures. Three biotrickling filters were run in parallel at temperatures ranging from 40 degrees C (mesophilic control) to 70 degrees C. The first phase involved treatment of methanol, for a 3-month run, and the second phase involved a 260-day run on the treatment of alpha-pinene. Methanol removal rates over 100 g m(-3) h(-1) where achieved at temperatures up to 70 degrees C. Alpha-pinene removal was achieved at temperatures up to 60 degrees C with optimal treatment occurring at 55 degrees C at rates up to 60 g m(-3) h(-1). The time for acclimation increased with increasing temperature and was longer for pinene than for methanol. Filter performance was also able to quickly recover from a shutdown period of up to 2 weeks due to the robustness of the microbial communities as determined by DNA fingerprinting analysis. The high-temperature communities treating methanol or pinene were more similar to each other than the mesophilic communities (i.e., 40 degrees C). The mesophilic methanol community had a high degree of functional redundancy, while the mesophilic pinene community was more unique and very distinct from the others. These results show that biofiltration at high temperatures is achievable and opens up a range of possibilities for applying biofiltration to hot gas streams.

The occurrence of cyanobacteria in pulp and paper waste-treatment systems.

Pulp and paper secondary waste-treatment systems in Brazil, Canada, New Zealand, and the U.S.A. contained dynamic cyanobacterial communities, some of which exceeded heterotrophic bacterial biomass. No other viable photoautotrophic populations were detected in the ponds. Regardless of geographical location, Oscillatoriales including Phormidium, Geitlerinema, and Pseudanabaena were the dominant taxa. As well, Chroococcus (Chroococcales) was an important genus in Brazil and New Zealand. The possible impact of cyanobacteria on waste-treatment efficiency deserves further study given their large biomass and diverse metabolic characteristics.

A comparison of the ability of forest and agricultural soils to mineralize chlorinated aromatic compounds.

Soils were sampled from two agricultural fields, two relatively pristine forests, and one suburban forest in Ontario, Canada. The ability of these soils to mineralize 2,4-dichlorophenoxyacetate, 3-chlorobenzoate, 4-chlorophenol, 2,4-dichlorophenol, pentachlorophenol, and atrazine was determined using 14C-labeled substrates. Direct pre-exposure was necessary before atrazine mineralization could be detected; however, it was not necessary for degradation of any of the other chemicals. 2,4-dichlorophenoxyacetate and pentachlorophenol mineralization was much higher in the agricultural soils relative to the pristine forest soils, but 3-chlorobenzoate and 2,4-dichlorophenol mineralization rates showed the opposite trend. Mineralization of 4-chlorophenol was about equivalent in all soils. Suburban forests soils were indistinguishable from agricultural soils with respect to their degradation of 2,4-dichlorophenoxyacetate and chlorobenzoate. Additionally, they were better able than any of the soils to withstand the toxic effects of pentachlorophenol. Pentachlorophenol mineralization was highly variable in the pristine forest soils, ranging from about 6 to 50%. Abiotic factors such as pH, soil type, and organic and moisture content did not account for these significant site differences. The selective forces responsible for these differences, and the possible differences in microbial populations are discussed.

Molecular analysis of bacterial isolates and total community DNA from kraft pulp mill effluent treatment systems.

Chloroaliphatics are major components of bleached kraft mill effluents. Gene probes and oligonucleotide primers were developed to monitor kraft pulp mill effluent treatment systems for the presence of key genes (dehalogenases) responsible for the dehalogenation of chloroaliphatic organics. The primers were used for polymerase chain reaction (PCR) analysis of genomic DNA extracted from dehalogenating bacterial isolates and from total community DNA extracted from water and sediments of mill effluent treatment system. PCR amplification with oligonucleotide primers designed from dhlB, encoding the haloacid dehalogenase from Xanthobacter autotrophicus, revealed the presence of dehalogenase genes in both aerated lagoons and stabilization basins. Similarly, positive results were obtained with mmoX primers designed from the soluble methane monooxygenase gene of Methylococcus capsulatus Bath. The haloacetate dehalogenase encoding gene (dehH2) from Moraxella sp. was typically not detected in mill effluent treatment systems unless the biomass was selectively enriched. DNA sequence analysis of several PCR fragaments revealed significant similarity to known dehalogenase amd methane monooxygenase genes. The results indicated a broad distribution of known dehalogenation genes and bacteria with chloroorganic-degrading potential in the mill effluent treatment systems.

High levels of endemicity of 3-chlorobenzoate-degrading soil bacteria.

Soils samples were obtained from pristine ecosystems in six regions on five continents. Two of the regions were boreal forests, and the other four were Mediterranean ecosystems. Twenty-four soil samples from each of four or five sites in each of the regions were enriched by using 3-chlorobenzoate (3CBA), and 3CBA mineralizers were isolated from most samples. These isolates were analyzed for the ability to mineralize 3CBA, and genotypes were determined with repetitive extragenic palindromic PCR genomic fingerprints and restriction digests of the 16S rRNA genes (amplified ribosomal DNA restriction analysis [ARDRA]). We found that our collection of 150 stable 3CBA-mineralizing isolates included 48 genotypes and 44 ARDRA types, which formed seven distinct clusters. The majority (91%) of the genotypes were unique to the sites from which they were isolated, and each genotype was found only in the region from which it was isolated. A total of 43 of the 44 ARDRA types were found in only one region. A few genotypes were repeatedly found in one region but not in any other continental region, suggesting that they are regionally endemic. A correlation between bacterial genotype and vegetative community was found for the South African samples. These results suggest that the ability to mineralize 3CBA is distributed among very diverse genotypes and that the genotypes are not globally dispersed.

Monoclonal antibodies to 2,4-dichlorophenol hydroxylase as probes for the 2,4-D-degradative phenotype.

Two different monoclonal antibodies (MAb) were raised against 2,4-dichlorophenol hydroxylase (DCP-hydroxylase) of Ralstonia eutropha JMP134 (pJP4), the second enzyme in the 2,4-D-degradative pathway of this bacterium. The utility of these antibodies in detecting and characterizing 2,4-D-degrading soil bacteria was investigated. One MAb (F6) reacted with DCP-hydroxylase from 27 out of 36 strains tested, while the other (MAb C3) reacted with only 17 isolates. When used with the colony blot technique, MAb F6 was useful for detecting cross-reacting strains on plates of pure cultures or of mixtures containing nondegraders even when 2,4-D degraders were outnumbered 60 to 1. 2,4-D-degrading strains could also be detected from plates spread with enrichment cultures but not from primary isolation plates spread from soil dilutions, presumably because the ratio of degraders to nondegraders was too low. Colonies of some strains that were very distantly related genetically, but produced functionally similar DCP-hydroxlase enzymes, were detected by MAb F6. This result suggests that MAbs could be useful for detecting functionally similar proteins expressed from tfdB analogs, even in the absence of detectable DNA homology between the genes encoding them.

Pristine environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria.

2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure. By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D. Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequencing and physiological properties revealed that one isolate from Hawaiian volcanic soil could be classified in the genus Variovorax (a member of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of similarity to those of the Bradyrhizobium group (a member of the alpha subdivision of the class Proteobacteria). All the isolates grow slowly on either nutrient media (0.1 x Bacto Peptone-tryptone-yeast extract-glucose [PTYG] or 0.1 x Luria broth [LB] medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than previously known 2,4-D degraders. Nutrient-rich media such as full-strength PTYG and LB medium did not allow their growth. PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligenes eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primers and Southern hybridization with pJP4-tfdA as a probe revealed that the isolate belonging to the genus Variovorax carried the tfdA gene. This gene was transmissible to A. eutrophus JMP228 carrying a plasmid with a mutant tfdA gene. The other five isolates did not appear to carry tfdA, and 2,4-D-specific alpha-ketoglutarate-dependent dioxygenase activity could not be detected in cell lysates. These results indicate that 2,4-D-degrading bacteria in pristine environments are slow-growing bacteria and that most of their phylogenies and catabolic genes differ from those of 2,4-D degraders typically isolated from agricultural soils or contaminated environments.

Pristine soils mineralize 3-chlorobenzoate and 2,4-dichlorophenoxyacetate via different microbial populations.

Biodegradation of two chlorinated aromatic compounds was found to be a common capability of the microorganisms found in the soils of undisturbed, pristine ecosystems. We used 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3CBA) as enrichment substrates to compare populations of degrading bacteria from six different regions making up two ecosystems. We collected soil samples from four Mediterranean (California, central Chile, the Cape region of South Africa, and southwestern Australia) and two boreal (northern Saskatchewan and northwestern Russia) ecosystems that had no direct exposure to pesticides or to human disturbance. Between 96 and 120 samples from each of the six regions were incubated with 50 ppm of [U-14C]2,4-D or [U-14C]3CBA. Soils from all regions samples mineralized both 2,4-D and 3CBA, but 3CBA was mineralized without a lag period, while 2,4-D was generally not mineralized until the second week. 3CBA degradative capabilities were more evenly distributed spatially than those for 2,4-D. The degradative capabilities of the soils were readily transferred to fresh liquid medium. 3CBA degraders were easily isolated from most soils. We recovered 610 strains that could release carbon dioxide from ring-labeled 3CBA. Of these, 144 strains released chloride and degraded over 80% of 1 mM 3CBA in 3 weeks or less. In contrast, only five 2,4-D degraders could be isolated, although a variety of methods were used in an attempt to culture the degraders. The differences in the distribution and culturability of the bacteria responsible for 3CBA and 2,4-D degradation in these ecosystems suggest that the two substrates are degraded by different populations. We also describe a 14C-based microtiter plate method that allows efficient screening of a large number of samples for biodegradation activity.

The phylogenetic distribution of a transposable dioxygenase from the Niagara River watershed.

Horizontal gene transfer in the Bacteria has been demonstrated to occur under natural conditions. The ecological impact of gene transfer events depends on the new genetic material being expressed in recipient organisms, and on natural selection processes operating on these recipients. The phylogenetic distribution of cbaAB genes for chlorobenzoate 3,4-(4,5)-dioxygenase, which are carried within Tn5271 on the IncP beta plasmid pBRC60, was investigated using isolates from freshwater microcosms and from the Niagara River watershed. The latter included isolates from surface water, groundwater and bioremediation reactor samples. The cbaAB genes have become integrated, through interspecific transfer, primarily into species of the beta Proteobacteria (44/48 isolates). Only four isolates, identified as Pseudomonas fluorescens (3/48) and Xanthomonas maltophilia (1/48), belonged to the gamma Proteobacteria, despite the observation that pBRC60 was capable of mobilizing these genes into a wide range of beta and gamma Proteobacteria in the laboratory. The natural host range correlated with the distribution of the meta-ring-fission pathway for metabolism of protocatechuates formed when the cbaAB genes were expressed (45/48 isolates). We proposed the hypothesis that natural selection has favoured recipients that successfully integrate the activity of the transferred dioxygenase with the conserved meta ring-fission pathway. The hypothesis was tested by transferring a plasmid construct containing the cbaAB genes into type strains representative of the beta and gamma Proteobacteria. The concept of applying mobile catabolic genes to probe the phylogenetic distribution of compatible degradative pathways is discussed.

2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes.

DNA from 32 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria from diverse locations was probed with the first three genes of the well-known 2,4-D degradation pathway found in Alcaligenes eutrophus JMP134(pJP4). The majority of strains did not show high levels of homology to the first three genes of the 2,4-D degradation pathway, tfdA, -B, and -C. Most strains showed combinations of tfdA-, B-, and C-like elements that exhibited various degrees of homology to the gene probes. Strains having the same genomic fingerprints (as determined by repetitive extragenic palindromic PCR) exhibited the same hybridization pattern regardless of the geographic origin of the strain, with the exception of a strain isolated from Puerto Rico. This strain had the same genomic fingerprint as that of numerous other strains in the collection but differed in its hybridization against the tfdA gene probe. Members of the beta subdivision of the Proteobacteria class, specifically Alcaligenes, Burkholderia, and Rhodoferax species, carried DNA fragments with 60% or more sequence similarity to tfdA of pJP4, and most carried fragments showing at least 60% homology to tfdB. However, many strains did not hybridize with tfdC, although they exhibited chlorocatechol dioxygenase activity. Members of the alpha subdivision of the Proteobacteria class, mostly of the genus Sphingomonas, did not hybridize to either tfdA or tfdC, but some hybridized at low stringency to tfdB. The data suggest that extensive interspecies transfer of a variety of homologous degradative genes has been involved in the evolution of 2,4-D-degrading bacteria.

A comparison of organochlorine removal from bleached kraft pulp and paper-mill effluents by dehalogenating Pseudomonas, Ancylobacter and Methylobacterium strains.

The relative importance of each of three dechlorinating species to overall organochlorine removal from bleached kraft-mill effluents (BKME) was assessed. Ancylobacter aquaticus A7, Pseudomonas P1, and Methylobacterium CP13, strains indigenous to a BKME treatment system, were tested for growth on chlorinated acetic acids and alcohols, and for adsorbable organic halogen (AOX) reduction in batch cultures of sterile BKME from three sources. A. aquaticus A7 exhibited the broadest substrate range, but could only affect significant AOX reduction in softwood effluents. Methylobacterium CP13 exhibited a limited substrate range, but was capable of removing significant amounts of AOX from both hardwood and softwood effluents. By contrast, Pseudomonas sp. P1 exhibited a limited substrate range and poor to negligible reductions in AOX levels from both effluent types. Mixed inocula of all three species combined and inocula of sludge from mill treatment systems removed as much AOX from softwood effluents as did pure populations of Methylobacterium CP13. When BKME was hydrolysed prior to AOX analysis, the subsequent estimates of recalcitrant, or non-hydrolysable, AOX levels were far less variable than their counterpart total AOX measures. It is suggested that this is a relevant and useful measure of AOX for biodegradation studies.