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In this study, we investigated the feasibility of anaerobic sewage treatment at extremely low temperatures (4 °C) using two reactor setups: upflow anaerobic sludge blanket reactors (UASB) without and with (AnMBRUASB(UF)) a membrane. Both reactors were inoculated with seeds derived from sediments that were putatively acclimatized to low temperatures. A preliminary batch trial showed that treatment is feasible with the removal of carbon coupled to methane and sulphide production. The reactors operated for 180 days at a hydraulic retention time of 3 days. After 40 days acclimation, both systems met the EU chemical oxygen demand (COD) effluent standard (

Quantitative Structure Biodegradation Relationships (QSBRs) are a tool to predict the biodegradability of chemicals. The objective of this work was to generate reliable biodegradation data for mono-aromatic chemicals in order to evaluate and verify previously developed QSBRs models. A robust biodegradation test method was developed to estimate specific substrate utilization rates, which were used as a proxy for biodegradation rates of chemicals in pure culture. Five representative mono-aromatic chemicals were selected that spanned a wide range of biodegradability. Aerobic biodegradation experiments were performed for each chemical in batch reactors seeded with known degraders. Chemical removal, degrader growth and CO2 production were monitored over time. Experimental data were interpreted using a full carbon mass balance model, and Monod kinetic parameters (Y, Ks, qmax and μmax) for each chemical were determined. In addition, stoichiometric equations for aerobic mineralization of the test chemicals were developed. The theoretically estimated biomass and CO2 yields were similar to those experimentally observed; 35% (s.d ± 8%) of the recovered substrate carbon was converted to biomass, and 65% (s.d ± 8%) was mineralised to CO2. Significant correlations were observed between the experimentally determined specific substrate utilization rates, as represented by qmax and qmax/Ks, at high and low substrate concentrations, respectively, and the first order biodegradation rate constants predicted by a previous QSBR study. Similarly, the correlation between qmax and selected molecular descriptors characterizing the chemicals structure in a previous QSBR study was also significant. These results suggest that QSBR models can be reliable and robust in prioritising chemical half-lives for regulatory screening purposes.

Growth and extensive urbanisation of the human population has been accompanied by increased manufacture and use of chemical compounds. To classify the fate and behaviour of these compounds in the environment, a series of international standardised biodegradation screening tests (BSTs) were developed over 30 years ago. In recent years, regulatory emphasis (e.g. REACH) has shifted from measuring biodegradation towards prioritisations based on chemical persistence. In their current guise, BSTs are ineffective as screens for persistence. The marine BST OECD 306 in particular is prone to high levels of variation and produces a large number of fails, many of which can be considered false negatives. An ECETOC funded two-day workshop of academia, industry and regulatory bodies was held in 2015 to discuss improvements to the marine BSTs based on previous research findings from the Cefic LRI ECO11 project and other foregoing studies. During this workshop, methodological improvements to the OECD 306 test were discussed, in addition to clarifying guidance on testing and interpretation of results obtained from marine BSTs (such as pass criteria, lag phases, freshwater read across and complex substances). Methodologically: (i) increasing bacterial cell concentrations to better represent the bacterial diversity inherent in the sampled environments; and (ii) increasing test durations to investigate extended lag phases observed in marine assessments, were recommended to be validated in a multi-institutional ring test.

This study aims to evaluate micropollutant occurrence and removal in a low-middle income country (LMIC) by investigating the occurrence of 28 chemicals from different classes (triclosan, 15 polycyclic aromatic hydrocarbons (PAHs), 4 estrogens and 8 polybrominated diphenyl ether (PBDE) congeners) in three technologically diverse full-scale Brazilian wastewater treatment plants (WWTPs). These chemicals were detected at concentrations similar to those reported in other low-middle income countries (LMICs) and high-income countries (HICs) (0.1–49 μg/L) indicating their widespread use globally and the need for more studies in LMICs that are typically characterized by relatively inadequate wastewater treatment barriers. Among the three different WWTPs investigated for removal of these chemicals, the least energy intensive system, waste stabilization ponds (WSPs), was the most effective (95–99%) compared to the activated sludge (79–94%), and Up-flow sludge blanket reactor (UASB) with trickling filters system (89–95%). These results highlight the potential of WSPs for micropollutant removal-especially in warm climates. However, the effluent from all three WWTP could pose a risk to aquatic organisms when discharged into the receiving waters as the effluent concentrations of triclosan, some estrogens, PAHs and BDE 209 were above European environmental quality standards (EQS) or predicted no effect concentration (PNEC values), indicating that receiving water bodies could benefit from further treatment. In combination, these results help to further understand prevailing concentrations of micropollutants globally and fate in current wastewater treatment systems.

Quorum sensing (QS), a microbial communication mechanism modulated by acyl homoserine lactone (AHL) molecules impacts biofilm formation in bioreactors. This study investigated the effects of temperature and immigration on AHL levels and biofouling in anaerobic membrane bioreactors. The hypothesis was that the immigrant microbial community would increase the AHL-mediated QS, thus stimulating biofouling and that low temperatures would exacerbate this. We observed that presence of immigrants, especially when exposed to low temperatures indeed increased AHL concentrations and fouling in the biofilms on the membranes. At low temperature, the concentrations of the main AHLs observed, N-dodecanoyl-L-homoserine lactone and N-decanoyl-L-homoserine lactone, were significantly higher in the biofilms than in the sludge and correlated significantly with the abundance of immigrant bacteria. Apparently low temperature, immigration and denser community structure in the biofilm stressed the communities, triggering AHL production and excretion. These insights into the social behaviour of reactor communities responding to low temperature and influx of immigrants have implications for biofouling control in bioreactors.

The effects of temperature reduction (from 35 °C to 20 °C) on nitrogen removal performance and microbial diversity of an anammox sequencing batch reactor were evaluated. The reactor was fed for 148 days with anaerobically pretreated municipal wastewater amended with nitrite. On average, removal efficiencies of ammonium and nitrite were high (96%) during the enrichment period and phases 1 (at 35 °C) and 2 (at 25 °C), and slightly decreased (to 90%) when the reactor was operated at 20 °C. Deep sequencing analysis revealed that microbial community structure changed with temperature decrease. Anammox bacteria (Ca. Brocadia and Ca. Anammoximicrobium) and denitrifiers (Burkholderiales, Myxococcales, Rhodocyclales, Xanthomonadales, and Pseudomonadales) were favoured when the temperature was lowered from 35 °C to 25 °C, while Anaerolineales and Clostridiales were negatively affected. The results support the feasibility of using the anammox process for mainstream nitrogen removal from anaerobically pretreated municipal wastewater at typical tropical temperatures.

Poor breakdown of lipids is a major barrier to the anaerobic treatment of domestic wastewater at low temperatures. Lipids are degraded by extracellular lipases that are produced by, as yet, unknown bacteria. We wished to know if the poor degradation of lipids at low temperatures is due to a lack of lipase or to a lack of lipase activity. We therefore conducted an assay of the lipase activity in biomass adapted to the treatment of settled domestic wastewater at 4, 8 and 15 °C and compared this with the activity of the same biomass at 37 °C. Lipase activity decreased with temperature and was un-measurable at 4 °C. When the lipase activity in the biomass was evaluated at 37 °C, the lipase activity was 21.7 ± 14.0% greater in the biomass adapted to 4 °C than in the biomass adapted to 15 °C. We conclude that enzymes are produced at low temperatures but that the activity at these temperatures is low. Settled and unsettled wastewaters were used as controls and we were surprised to see lipolytic activity in the unsettled wastewaters at low temperatures. The activity was too high to be accounted for by the presence of biological detergents and we infer that is caused by bacteria naturally present in domestic wastewater. The presence of lipolytic activity at low temperatures was associated with the presence of members of the genera Trichococcus and Devosia, and the families Caldilineaceae and Bacteroidaceae.

This study evaluated the microbiological safety of the water distribution system of a city in the state of Minas Gerais (Brazil), population 120,000 inhabitants. During the study, the city suffered a severe drought that had a significant impact on water availability and quality in the river that supplies water to the city. Samples (2 liters) were collected from the distribution system over a period of six months, which included wet and dry months, from three points: the point with the lowest altitude in the distribution network, the farthest point from the water treatment works, and an intermediate point. Free chlorine was measured in situ using a Hach kit. DNA was extracted using a FastDNA Spin Kit Soil (Qbiogene). Advanced sequencing techniques (Ion Torrent) were used to identify and quantify the relative abundance of potentially pathogenic bacteria present in the samples. Coliforms and Escherichia coli, indicators currently used worldwide to assess microbiological safety of drinking water, were measured on all samples using an enzyme substrate method (ONPG-MUG Colilert®). Next generation sequencing results retrieved 16SrRNA sequences of E. coli and some potentially pathogenic bacteria, even in the presence of free chlorine. Operational taxonomic units related to pathogenic bacteria were present in all samples from the drinking water distribution system (DWS) and, in general, at high relative abundance (up to 5%). A total of 19 species related to bacterial pathogens were detected. Inadequate operational practices that could affect the microbiological safety of the DWS were identified and discussed. The current paper is the first to evaluate the community of potentially pathogenic bacteria in a real DWS.

The removal of β-estradiol (E2) and α-ethinylestradiol (EE2) in biological wastewater treatment plants (WWTP) would need to be improved in order to comply with prospective Environmental Quality Standards (EQS) of 0.4 and 0.035 ng.L−1 respectively. The effluent concentration of a micropollutant in an activated sludge process is a function of the removal rate, the hydraulic retention time (HRT) and the flow pattern, which is usually overlooked. In order to better understand this aspect, we carried out tracer studies in eight WWTPs in the UK and found that relatively modest changes in aeration tanks would translate into tangible improvements in their flow pattern. We further evaluated the degradation rates for E1 (estrone), E2, E3 (estriol) and EE2 in each WWTP and we estimated that the modification of the flow pattern would be sufficient to place effluent concentrations of E2 (23.2 L∙gVSS−1∙d−1

Quantitative PCR (qPCR) and next generation sequencing (NGS) are nucleic acid based microbiology techniques that provide new insights into drinking water quality, but considerable uncertainty remains around their correct interpretation. We noticed the presence of bacterial DNA from various putative pathogens, including from faecal indicator bacteria (FIB), in disinfected water, when culturable FIB were absent. To understand these observations better we studied the effect of chlorination on conventional and DNA based microbial water quality assessments. Surface water chlorination reduced plate counts for various FIB by up to >6 log units, intact cell counts by flow cytometry by 3.3 log units, and 16S rRNA gene copies by qPCR by 1.5 and 1.6 log units for total bacteria and total coliforms, respectively. Nanopore sequencing of 16S rRNA amplicons with the portable MinION device revealed the DNA from several families containing putative pathogens appeared to be more resistant than that of other bacteria to degradation by chlorine disinfection. For instance, 16S rRNA genes assigned to the Enterobacteriaceae family, members of which are mostly the target of coliform tests, increased in relative abundance from 0.001 ± 0.0002% to 0.0036 ± 0.003% after chlorine treatment. Hence, metagenomic drinking water data needs to be interpreted with caution. Plate counts and flow cytometry in combination with DNA based analysis provide more robust insight than NGS or qPCR alone.

Currently available OECD biodegradation screening tests (BSTs) are not particularly suited for persistence screening. Their duration can be much less than international half-life thresholds for persistence and they are variable and stringent, therefore prone to false negatives. The present study extended test durations beyond 28 days and increased biomass concentrations for marine BSTs to better represent the microbial diversity inherent in the sampled environment. For this so-called environmentally relevant BST (erBST) marine cell concentrations were nominally increased 100-fold by tangential flow filtration. The marine erBST was validated against a standard BST using five 14C labeled reference compounds with a range of biodegradation potentials (aniline, 4-fluorophenol, 4-nitrophenol, 4-chloroaniline and pentachlorophenol) in a modified OECD 301B test. A full mass balance was collated to follow chemical fate in the tests. The erBST was more accurate and less variable than the comparator BST in assigning the reference compounds to their expected biodegradation classifications (non-persistent or potentially persistent). According to the REACH non-persistence criterion of ≥60% biodegradation over 60 days, the erBST correctly classified 60% of chemical replicates according to their expected biodegradation classification and had a coefficient of variation of 21% between replicates. In contrast, the BST correctly assessed 40% of reference chemicals in regards to their expected biodegradation classification with a coefficient of variation of 36%. All non-persistent chemicals showed increased degradation in the erBST, except for 4-chloroaniline, which did not degrade in either BST or erBST. Both tests showed no false positive results, correctly classifying the negative control pentachlorophenol as potentially persistent. Next, it is recommended to further validate the marine erBST in an inter-laboratory study incorporating different seawater sources to fully assess its variability and reliability.

A natural Mn oxide (NMO) waste was assessed as an in situ remediation amendment for Pb contaminated sites. The viability of this was investigated using a 10 month lysimeter trial, wherein a historically Pb contaminated soil was amended with a 10% by weight model NMO. The model NMO was found to have a large Pb adsorption capacity (qmax 346±14 mg g(-1)). However, due to the heterogeneous nature of the Pb contamination in the soils (3650.54-9299.79 mg kg(-1)), no treatment related difference in Pb via geochemistry could be detected. To overcome difficulties in traditional geochemical techniques due to pollutant heterogeneity we present a new method for unequivocally proving metal sorption to in situ remediation amendments. The method combines two spectroscopic techniques; namely electron probe microanalysis (EPMA) and X-ray photoelectron spectroscopy (XPS). Using this we showed Pb immobilisation on NMO, which were Pb free prior to their addition to the soils. Amendment of the soil with exogenous Mn oxide had no effect on microbial functioning, nor did it perturb the composition of the dominant phyla. We conclude that NMOs show excellent potential as remediation amendments.

Peri-urban aquacultures produce nutritious food in proximity to markets, but poor surface water quality in rapidly expanding megacities threatens their success in emerging economies. Our study compared, for a wide range of parameters, water quality downstream of Bangkok with aquaculture regulations and standards. For parameters not meeting those requirements, we sought to establish whether aquaculture practice or external factors were responsible. We applied conventional and advanced methods, including micropollutant analysis, genetic markers, and 16S rRNA amplicon sequencing, to investigate three family-owned aquacultures spanning extensive, semi-intensive and intensive practices. Canals draining the city of Bangkok did not meet quality standards for water to be used in aquaculture, and were sources for faecal coliforms, Bacteriodes, Prevotella, Human E. coli, tetracycline resistance genes, and nitrogen into the aquaculture ponds. Because of these inputs, aquacultures suffered algae blooms, with and without fertilizer and feed addition to the ponds. The aquacultures were sources of salinity and the herbicide diuron into the canals. Diuron was detectable in shrimp, but not at a level of concern to human health. Given the extent and nature of pollution, peri-urban water policy should prioritize charging for urban wastewater treatment over water fees for small-scale agricultural users. The extensive aquaculture attenuated per year an estimated twenty population equivalents of nitrogen pollution and trillions of faecal coliform bacteria inputs from the canal. Extensive aquacultures could thus contribute to peri-urban blue-green infrastructures providing ecosystem services to the urban population such as flood risk management, food production and water pollution attenuation., Graphical abstract Unlabelled Image, Highlights • Extensive, semi-intensive and intensive aquacultures near Bangkok were investigated. • Aquaculture water quality was severely affected by urban pollution in the canals. • Aquacultures released herbicides/algaecides into canals. • Extensive aquacultures attenuated faecal pollution inputs from the canals. • Extensive aquaculture could be integrated in blue-green infrastructures.

Two strategies exist for seeding low-temperature anaerobic reactors: the use of specialist psychrophilic biomass or mesophilic bioreactor sludge acclimated to low temperature. We sought to determine the low-temperature limitation of anaerobic sludge from a bioreactor acclimated to UK temperatures (

Temperature is the bottleneck for the anaerobic treatment of domestic wastewater in temperate climates. Most previous attempts to achieve anaerobic treatment at low temperatures have attempted to acclimatize mesophilic sludge and have failed at temperatures below 10–13 °C. We describe an alternative approach using communities from environments that have been exposed to low temperatures over evolutionary time-scales as seed for such reactors. Batch reactors were inoculated with a mixture of soils and sediments from the high Arctic and an Alpine lake to treat UV-sterilized raw domestic wastewater at 4, 8 and 15 °C. To evaluate the intrinsic treatment capacity of the bacteria the specific rates of methanogenesis and hydrolysis were evaluated. Specific methanogenic activity at 4, 8 and 15 °C was 6.3, 7.6 and 10.3 fmol CH4 cell−1day−1 respectively. Specific putative hydrolysis rates were 76.2, 186.6 and 251.9 fgrams COD cell−1day−1. Hydrolysis was twice as temperature sensitive as methanogenesis (Q10: 4.62 and 1.57 respectively). The specific rates are over ten times higher than we have previously observed in microcosms fed with settled wastewater at the same temperatures. The results imply that inoculating reactors with cold-adapted communities is a promising way to develop biomass capable of treating anaerobic wastewater treatment at low temperatures whilst achieving an effluent that conforms to the EC Directive COD standards. Large-scale reactors are feasible if satisfactory cell concentrations can be achieved.

Biological treatment processes are "complex systems" where many different kinds of microbes grow and interact in a dynamic manner. Understanding the relationship between microbial diversity and bioreactor performance could facilitate the optimisation of bioreactor design and enable the solution of bioreactor-related problems. However, systematic studies of the effects of operating variables on microbial diversity and reactor performance are rare. In this study, we determined the effects of different operating conditions and system configurations on the performance of laboratory-scale activated sludge reactors and microbial diversity, based on experiments designed using the factorial design approach. We found that the overall system performance and the diversity of the microbial communities in the reactors were affected by changes in the operating parameters. However, the relationship between diversity and performance was sometimes counterintuitive, as increases in system performance were not always associated with increased community diversity. Reactor configuration and addition of soil had the biggest effects on reactor performance, while the effects of organic loading rates and feed composition were less marked. Of all these parameters, reactor configuration was the only one that had a consistent effect on reactor community diversity.

Recent studies using quantitative fluorescence in situ hybridization (FISH) have supported the principle that there are mycolata concentration thresholds, above which foaming is likely to occur. In this study, we surveyed 14 wastewater treatment plants (WWTPs) in the UK, using quantitative FISH, to establish that the principle of a mycolata threshold (2 x 10(6) mycolata cells ml(-1) mixed liquor suspended solids) is an empirical though widely held value. In addition, we designed, optimized and applied probes for members of the less hydrophobic mycolata genera Corynebacterium and Dietzia, to show that these organisms dominated the mycolata populations in two non-foaming WWTPs where the mycolata concentrations were above the threshold value. We propose that the mycolata threshold value is only applicable to hydrophobic members of the mycolata.

The microbial mechanisms of carbon removal in subsurface flow wetlands were studied, in one wetland with plants and one without plants. Particular emphasis was given to the influence of plants in the treatment process. Wetlands without plants showed higher carbon removal than those with plants, 63% and 51%, respectively (p

In a previous study, we showed that there was a significant increase in mycolata numbers associated with foaming events at a wastewater treatment site possessing three activated sludge plants. In this paper, we demonstrate that branched filamentous mycolata were a minor proportion of the mycolata morphotypes present in those activated sludge plants, accounting for less than 21% of the mycolata population in the mixed liquor and foam samples examined. In most samples examined, the number of filamentous mycolata was negligible compared to the number of other mycolata morphotypes present. Furthermore, filamentous mycolata did not contribute to any of the significant differences in mycolata concentration observed between foaming and non-foaming situations (P

The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.

A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.

Bioremediation and activated carbon (AC) amendment were compared as remediation strategies for sediment from the River Tyne containing 16.4 +/- 7.3 microg/g polycyclic aromatic hydrocarbons (PAHs) and approximately 5% coal particles by total dry sediment weight. Unamended, nutrient amended (biostimulated) and nutrient and Pseudomonas putida amended (bioaugmented) sediment microcosms failed to show a significant decrease in total sediment PAH concentrations over a one month period. Polyethylene passive (PE) samplers were embedded for 21 days in these sediment microcosms in order to measure the available portion of PAHs and accumulated 4.70 +/- 0.25, 12.43 +/- 1.78, and 23.49 +/- 2.73 microg PAHs/g PE from the unamended, biostimulated, and bioaugmented microcosms, respectively. Higher PAH uptake by PE samplers in biostimulated and bioaugmented microcosms coincided with slower degradation of spiked phenanthrene in sediment-free filtrate from these microcosms compared to filtrate from the unamended microcosms. Microbial community analysis revealed changes in the bacterial community directly following the addition of nutrients, but the added P. putida community failed to establish itself. Addition of 2% by dry sediment weight activated carbon reduced PAH uptake by PE samplers to 0.28 +/- 0.01 microg PAHs/g PE, a greater than 90% reduction compared to the unamended microcosms.

This study investigated the formation of the microbial communities in two horizontal subsurface-flow laboratory-scale constructed wetlands, one planted and the other one unplanted. The abundance of the predominant functional groups (Archaea, Bacteria and sulphate-reducing bacteria) was determined using fluorescence in situ hybridization and the diversity and community structure of those functional groups were analysed using denaturing gradient gel electrophoresis. The numbers of Archaea, Bacteria and sulphate-reducing bacteria were indistinguishable in both reactors (P=0.99, 0.80 and 0.55, respectively). The microbial communities in both wetlands were typically no more similar than if they had been randomly assembled from a common source community. Plants did not appear to exert a strong effect on the structure of the microbial communities in the horizontal subsurface-flow constructed wetlands (HSCWs) studied in this investigation.

We wished to discover if we could gain greater insights into how biological treatment plants function by contrasting the presence and activity of the most abundant Bacteria in plug flow and completely mixed activated sludge plants. Presence was assessed by amplifying 16S rRNA gene fragments (using PCR) and activity by amplifying native 16S rRNA, using reverse-transcriptase PCR (RT-PCR), using Bacteria -specific primers. The amplified sequences were compared using denaturing gradient gel electrophoresis (DGGE). The plug flow plant exhibited a strong physico-chemical gradient with an initial anoxic zone, whilst the two completely mixed reactors did not. Similarities were observed between the profile of the banding pattern for presence and activity. However, in the plug flow reactor one prominent band was detected in the active population (16S rRNA) but was absent from the corresponding profile of the 16S rRNA gene. Sequencing of this band revealed its identity as a Nitrosomonas -like sequence. The intensity of the 16S rRNA sequenced varied along the physico-chemical gradient of the plug-flow reactor in a manner that coincided with the growth of ammonia-oxidising bacteria (AOB) and the loss of ammonia. This band was also absent from the completely mixed reactors, although significant numbers of AOB were detected in all systems (∼10 6 –10 8 cells ml −1 ) by fluorescence in situ hybridisation (FISH). An abundant and highly active AOB population was present in the anoxic zone of the plug-flow reactor where up to 60% of the total ammonia was removed. An examination of nitrogen removal/production rates, together with the above data, reveal that complex nitrogen removal processes occur in this system. These data also enabled the calculation of a specific in situ growth rate for the AOB as 0.12 h −1 .

Isolates from activated sludge foam were provisionally assigned to the genera Gordona and Tsukamurella on the basis of colony morphology and pigmentation. Representatives of the first group were compared with marker strains of validly described species of Gordona by using Curie-point pyrolysis mass spectrometry. Most of the isolates fell into a number of taxa which were equated with groups of marker strains which corresponded to the known Gordona species. In a corresponding experiment the activated sludge isolates, which were provisionally labelled “Tsukamurella spumae” formed a group which was well separated from the marker strains of the genus. Representatives of the isolates were found to have chemical properties consistent with their classification in the genus Tsukamurella. 16S rDNA sequence data, when taken with previous DNA:DNA relatedness results, suggest that Tsukamurella paurometabola is heterogeneous. Similarly, the sequence data provide some evidence that “Tsukamurella spumae” may merit recognition as a novel species.

OECD ready biodegradability tests have been central to understanding the biodegradation of chemicals from a regulatory perspective for many decades. They are not fit for contemporary prioritisation of chemicals based on persistence, however, due to the low concentration of inocula used, short duration and high variability between tests. Two OECD standard inoculum pretreatment methods (settlement and filtration) were investigated to observe their effect on the probability of biodegradation and associated changes in bacterial community structure and diversity of inocula sourced from the activated sludge process of wastewater treatment plants. Both settlement and filtration were shown to dramatically and significantly reduce the probability and increase the variability of biodegradation of 4-nitrophenol compared to the use of unprocessed inocula. These differences were associated with a significant hundred-fold reduction in cell numbers and solids content and a significant shift in bacterial community structure that was sometimes accompanied by significant reductions in detectable operational taxonomic unit richness and evenness. The natural variation (between different environments) and variation due to differential selection of bacterial communities (by different pretreatment methods) is offered as an explanation for the historical high variability in standard OECD ready biodegradability tests. Electronic supplementary material The online version of this article (doi:10.1007/s11356-013-2064-4) contains supplementary material, which is available to authorized users.