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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 (

Temperature is a key factor that influences chemical biotransformation potential and rates, on which exposure and fate models rely to predict the environmental (micro)pollutant fate. Arrhenius-based models are currently implemented in environmental exposure assessment to adapt biotransformation rates to actual temperatures, assuming validity in the 0-30 °C range. However, evidence on how temperature shifts affect the physicochemical and microbial features in biological systems is scarce, questioning the validity of the existing modeling approaches. In this work, laboratory-scale batch assays were designed to investigate how a mixed microbial community responds to short-term temperature shifts, and how this impacts its ability to biotransform a range of structurally diverse micropollutants. Our results revealed three distinct kinetic responses at temperatures above 20 °C, mostly deviating from the classic Arrhenius-type behavior. Micropollutants with similar temperature responses appeared to undergo mostly similar initial biotransformation reactions, with substitution-type reactions maintaining Arrhenius-type behavior up to higher temperatures than oxidation-type reactions. Above 20 °C, the microbial community also showed marked shifts in both composition and activity, which mostly correlated with the observed deviations from Arrhenius-type behavior, with compositional changes becoming a more relevant factor in biotransformations catalyzed by more specific enzymes (e.g., oxidation reactions). Our findings underline the need to re-examine and further develop current environmental fate models by integrating biological aspects, to improve accuracy in predicting the environmental fate of micropollutants.

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17β-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol.

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.

The use of activated carbon (AC) amendments to reduce exposure risks for hydrophobic contaminants like polycyclic aromatic hydrocarbons (PAHs) by adsorption is an innovative soil remediation approach. However, AC amendment side-effects on the pollution biodegradation are poorly understood. This study assessed for optimized nutrient ratio conditions, effects of 5% soil wet weight AC amendments, if added from the beginning or after five months, on the outcomes of one year of crude oil polluted soil bioremediation. CO2, residual hydrocarbon concentrations and microbial community structure analysis revealed how AC amendment hindered crude oil biodegradation much more, if added from the start, as compared to after 5 months, i.e. after the initial phase of biodegradation. Putative crude oil degrading microorganisms from the genera Marinobacter, Parvibaculum, Salinibacterium, Muricauda, and Alcanivorax were more sensitive to the AC amendment than those from the genus Rhodococcus. Rhodococci possess hydrophobic cell walls which may enable them to accumulate hydrocarbons in the AC amended soil, despite of their reduced availability. AC amendment from the start had the highest alkane and total US EPA PAH residues, but was more effective than one year of bioremediation, with or without AC amendment after 5 months, in reducing PAH availability in the soil.

The removal mechanisms and extent of degradation of 28 chemicals (triclosan, fifteen polycyclic aromatic hydrocarbons, four estrogens, and eight polybrominated diphenyl ether congeners) in different biological treatment systems [activated sludge, up-flow anaerobic sludge blanket reactor (UASB) and waste stabilization pond (WSP)] was investigated to provide insights into the limits of engineered biological treatment systems. This was done through degradation experiments with inhibition and abiotic controls in static reactors under aerobic and anaerobic conditions. Estrogens showed higher first order degradation rates (0.1129 h−1) under aerobic conditions with activated sludge inocula followed by low molecular weight (LMW) PAHs (0.0171 h−1), triclosan (0.0072 h−1), middle (MMW) (0.0054 h−1) and high molecular weight PAHs (HMW) (0.0033 h−1). The same trend was observed under aerobic conditions with a facultative inoculum from a WSP, although at a much slower rate. Biodegradation was the major removal mechanism for these chemicals in the activated sludge and WSP WWTPs surveyed. Photodegradation of these chemicals was also observed and varied across the group of chemicals (estrogens (light rate = 0.4296 d−1; dark = 0.3900 d−1) degraded faster under light conditions while reverse was the case for triclosan (light rate = 0.0566 d−1; dark = 0.1752 d−1). Additionally, all the chemicals were resistant to anaerobic degradation with UASB sludge, which implies that their removal in the UASB of the surveyed WWTP was most likely via sorption onto solids. Importantly, the first order degradation rate determined in this study was used to estimate predicted effluent concentrations (PECs). The PECs showed good agreement with the measured effluent concentrations from a previous study for these treatment systems.

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.

MotivationCodon optimisation, the process of adapting the codon composition of a coding sequence, is often used in synthetic biology to increase expression of a heterologous protein. Recently, a number of synthetic biology approaches that allow synthetic constructs to be deployed in multiple organisms have been published. However, so far, design tools for codon optimisation have not been updated to reflect these new approaches.ApproachWe designed an evolutionary algorithm (EA) to design coding sequences (CDSs) that encode a target protein for one or more target organisms, based on the Chimera average repetitive substring (ARS) metric — a correlate of gene expression. A parameter scan was then used to find optimal parameter sets. Using the optimal parameter sets, three heterologous proteins were repeatedly optimised Bacillus subtilis 168 and Escherichia coli MG1655. The ARS scores of the resulting sequences were compared to the ARS scores of coding sequences that had been optimised for each organism individually (using Chimera Map).ResultsWe demonstrate that an EA is a valid approach to optimising a coding sequence for multiple organisms at once; both crossover and mutation operators were shown to be necessary for the best performance. In some scenarios, the EA generated CDSs that had higher ARS scores than CDSs optimised for the individual organisms, suggesting that the EA exploits the CDS design space in a way that Chimera Map does not.Availability and implementationThe implementation of the EA, with instructions, is available on GitHub: https://github.com/intbio-ncl/chimera_evolve.

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.

Waste stabilisation ponds (WSPs) are widely used across the world as a passive wastewater treatment for domestic wastewaters, but little is known about their ecology, especially their phototrophic communities. This study uses molecular methods and flow cytometry to assess the cyanobacterial and eukaryotic communities longitudinally throughout two systems, one treating domestic wastewater and the other mixed industrial/domestic wastewaters. More variation was seen between the systems than between different stages in the treatment processes for both eukaryotic and cyanobacterial communities. Chlorella species and Planktophrix cyanobacteria dominated both treatment systems. Arthrospira cyanobacteria were detected only in the industrial/domestic system. The balance between non-photosynthetic and photosynthetic organisms is rarely considered, though both play vital roles in WSP functioning. Flow cytometry showed that the facultative and first maturation pond in the industrial system contained a lower proportion of photosynthetic organisms compared to the domestic system. This is reflected in the species richness data and low dissolved oxygen levels detected. All data indicated that both systems are significantly different from one another and that variation longitudinally throughout the systems is lower. A more systematic study is needed to determine if it is the wastewater source rather than the initial inoculum that drives community composition.

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.

We studied the effects of two percent by weight activated carbon versus biochar amendments in 93 cm long sand columns on the biofiltration of petroleum vapours released by a non-aqueous phase liquid (NAPL) source. Activated carbon greatly enhanced, whereas biochar slightly reduced, the biofiltration of volatile petroleum hydrocarbons (VPHs) over 430 days. Sorbent amendment benefitted the VPH biofiltration by retarding breakthrough during the biodegradation lag phase. Subsequently, sorbent amendment briefly reduced the mineralization of petroleum hydrocarbons by limiting their bioavailability. During the last and longest study period, when conditions became less supportive of microbial growth, because of inorganic nutrient scarcity, the sorbents again improved the pollution attenuation by preventing the degrading microorganisms from being overloaded with VPHs. A 16S rRNA gene based analysis showed sorbent amendment effects on soil microbial communities. Nocardioidaceae benefitted the most from petroleum hydrocarbons in activated carbon amended soil, whereas Pseudomonadacea predominated in unamended soil. Whilst the degrading microorganisms were overloaded with VPHs in the unamended soil, the reduced mobility and bioavailability of VPHs in the activated carbon amended soil led to the emergence of communities with higher specific substrate affinity, which removed bioavailable VPHs effectively at low concentrations. A numerical pollutant fate model reproduced these experimental observations by considering sorption effects on the pollutant migration and bioavailability for growth of VPH degrading biomass, which is limited by a maximum soil biomass carrying capacity. Activated carbon was a much stronger sorbent for VPHs than biochar, which explained the diverging effects of the two sorbents in this study.

Nucleic acid based techniques, such as quantitative PCR (qPCR) and next generation sequencing (NGS), provide new insights into microbial water quality, but considerable uncertainty remains around their correct interpretation. We demonstrate, for different water sources in informal settlements in the Kathmandu Valley, Nepal, significant Spearman rank correlations between conventional and molecular microbiology methods that indicate faecal contamination. At family and genera level, 16S rRNA amplicon sequencing results obtained with the low-cost, portable next generation sequencer MinION from Oxford Nanopore Technologies had significant Spearman rank correlations with Illumina MiSeq sequencing results. However, method validation by amplicon sequencing of a MOCK microbial community revealed the need to ascertain MinION sequencing results for putative pathogens at species level with complementary qPCR assays. Vibrio cholerae hazards were poorly associated with plate count faecal coliforms, but flagged up by the MinION screening method, and confirmed by a qPCR assay. Plate counting methods remain important to assess viability of faecal coliforms in disinfected water sources. We outline a systematic approach for data collection and interpretation of such complementary results. In the Kathmandu Valley, there is high variability of water quality from different sources, including for treated water samples, illustrating the importance of disinfection at the point of use.

Anaerobic digestate and biochar are by-products of the biogasification and pyrolysis of agricultural wastes. This study tested the hypothesis that combined application of anaerobic pig/cattle manure digestate and coconut husk (CH) biochar can improve soil nutrient conditions, whilst minimizing atmospheric and groundwater pollution risks. Microcosms simulated digestate application to agricultural soil with and without CH biochar. Ammonia volatilization and nutrient leaching were quantified after simulated heavy rainfalls. Archaeal and bacterial community and abundance changes in soils were quantified via next generation sequencing and qPCR of 16S rRNA genes. Nitrifying bacteria were additionally quantified by qPCR of functional genes. It was found that CH biochar retarded nitrate leaching via slower nitrification in digestate-amended soil. CH biochar reduced both nitrifying archaea and bacteria abundance in soil by 71–83 percent in the top 4 cm soil layer and 66–80 percent in the deeper soil layer one month after the digestate application. Methanotroph abundances were similarly reduced in the CH biochar amended soils. These findings demonstrate combined benefits of anaerobic digestate and CH biochar application which are relevant for the development of a more circular rural economy with waste minimization, renewable energy production, nutrient recycling and reduced water pollution from agricultural land., Graphical abstract Image 1, Highlights • Combined benefits of digestate and biochar application to soil were studied. • Microbial community response was monitored via next generation sequencing and qPCR. • Coconut husk biochar amendment slightly enhanced nutrient adsorption. • Biochar reduced nitrifier abundance and nitrification in digestate-amended soil. • Nutrients from biowastes were recycled with reduced groundwater pollution risks., Capsule: Chemical and molecular microbiology measurements elucidated biochar and anaerobic digestate effects on nutrient retention and nitrification in soil, and groundwater pollution risks.

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.

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 (

We tested the hypothesis that the biodegradation of volatile petroleum hydrocarbons (VPHs) in aerobic sandy soil is affected by the blending with 10 percent ethanol (E10) or 20 percent biodiesel (B20). When inorganic nutrients were scarce, competition between biofuel and VPH degraders temporarily slowed monoaromatic hydrocarbon degradation. Ethanol had a bigger impact than biodiesel, reflecting the relative ease of ethanol compared to methyl ester biodegradation. Denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA genes revealed that each fuel mixture selected for a distinct bacterial community, each dominated by Pseudomonas spp. Despite lasting impacts on soil bacterial ecology, the overall effects on VHP biodegradation were minor, and average biomass yields were comparable between fuel types, ranging from 0.40 ± 0.16 to 0.51 ± 0.22 g of biomass carbon per gram of fuel carbon degraded. Inorganic nutrient availability had a greater impact on petroleum hydrocarbon biodegradation than fuel composition.

Production of hydrogen sulphide by sulphate reducing bacteria (SRB) is a serious problem in anaerobic wastewater treatment, because it causes corrosion and reduces the value of methane in the biogas produced. The surface and adhesion characteristics of SRB as measured through the zeta potential are different from those of methanogens. We therefore tested the hypothesis that by choosing a carrier material with the proper surface characteristics (zeta potential) it should be possible to selectively immobilize methanogens while excluding SRB. In a series of batch tests with different support materials complete elimination of SRB was obtained on supports made of nylon, a result in line with our original hypothesis. Maximum preferential immobilization of methanogens was obtained at a temperature of 37 °C with media containing volatile fatty acids (VFA) as the carbon source. Preferential immobilization of methanogens was achieved in an anaerobic fluidized bed reactor (AFBR) with nylon granules as support and in an anaerobic rotating biological contactor (ARBC) with an acrylic disc as support. H2S-free biogas containing 63% and 38% methane were obtained from the AFBR and ARBC, respectively.

Rieske non-heme iron oxygenases enzymes have been widely studied, as they catalyse essential reactions initiating the bacterial degradation of organic compounds, for instance aromatic hydrocarbons. The genes encoding these enzymes offer a potential target for studying aromatic hydrocarbon-degrading organisms in the environment. However, previously reported primer sets that target dioxygenase gene sequences or the common conserved Rieske centre of aromatics dioxygenases have limited specificity and/or target non-dioxygenase genes. In this work, an extensive database of dioxygenase α-subunit gene sequences was constructed, and primer sets targeting the conserved Rieske centre were developed. The high specificity of the primers was confirmed by polymerase chain reaction analysis, agarose gel electrophoresis and sequencing. Quantitative polymerase chain reaction (qPCR) assays were also developed and optimized, following MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments). Comparison of the qPCR quantification of dioxygenases in spiked sediment samples and in pure cultures demonstrated an underestimation of the Ct value, and the requirement for a correction factor at gene abundances below 108 gene copies per g of sediment. Externally validated qPCR provides a valuable tool to monitor aromatic hydrocarbon degrader population abundances at contaminated sites., Our study aimed to re-evaluate the phylogeny of Rieske non-heme iron dioxygenases using only retrieved primary nucleic acid sequences for the development of quantitative real-time PCR primers., Graphical Abstract Figure. Our study aimed to re-evaluate the phylogeny of Rieske non-heme iron dioxygenases using only retrieved primary nucleic acid sequences for the development of quantitative real-time PCR primers.

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

Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase ( amoA ) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.

Society׳s reliance upon chemicals over the last few decades has led to their increased production, application and release into the environment. Determination of chemical persistence is crucial for risk assessment and management of chemicals. Current established OECD biodegradation guidelines enable testing of chemicals under laboratory conditions but with an incomplete consideration of factors that can impact on chemical persistence in the environment. The suite of OECD biodegradation tests do not characterise microbial inoculum and often provide little insight into pathways of degradation. The present review considers limitations with the current OECD biodegradation tests and highlights novel scientific approaches to chemical fate studies. We demonstrate how the incorporation of molecular microbial ecology methods (i.e., ‘omics’) may improve the underlying mechanistic understanding of biodegradation processes, and enable better extrapolation of data from laboratory based test systems to the relevant environment, which would potentially improve chemical risk assessment and decision making. We outline future challenges for relevant stakeholders to modernise OECD biodegradation tests and put the ‘bio’ back into biodegradation.

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods ( P << 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m 3 completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 × 10 6 cells ml −1 or 4 × 10 12 cells m −2 . We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.

We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.

Views of community assembly have traditionally been based on the contrasting perspectives of the deterministic niche paradigm and stochastic neutral models. This study sought to determine if we could use empirical interventions conceived from a niche and neutral perspective to change the diversity and evenness of the microbial community within a reactor treating wastewater and to see if there was any associated change in the removal of endocrine disrupting compounds (EDCs).The systematic removal EDCs and micropollutants from biological treatment systems is a major challenge for environmental engineers. We manipulated pairs of bioreactors in an experiment in which niche (temporal variation in resource concentration and resource complexity) and neutral (community size and immigration) attributes were changed and the effect on the detectable diversity and the removal of steroidal oestrogens was evaluated. The effects of manipulations on diversity suggested that both niche and neutral processes are important in community assembly. We found that temporal variation in environmental conditions increased diversity but resource complexity did not. Larger communities had greater diversity but attempting to increase immigration by adding soil had the opposite effect. The effects of the manipulations on EDC removal efficiency were complex. Decreases in diversity, which were associated with a decrease in evenness, were associated with an increase in EDC removal. A simple generalised neutral model (calibrated with parameters typical of wastewater treatment plants) showed that decreases in diversity should lead to the increase in abundance of some ostensibly taxa rare. We conclude that neither niche and neutral perspectives nor the effect of diversity on putative rare functions can be properly understood by naïve qualitative observations. Instead, the relative importance of the key microbial mechanisms must be determined and, ideally, expressed mathematically.

Large numbers of mycolic acid-containing actinomycetes were isolated from foam and scum samples taken from three activated-sludge sewage-treatment plants using several selective isolation media. Organisms presumptively identified as gordonae formed the dominant population in all of the samples. A representative set of these strains have chemical properties consistent with their classification in the genusGordona. Forty-eight of theGordona strains were compared through 165 unit characters with the type strains of validly described species ofGordona. The resultant data were examined using the Jaccard and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. The numerical classification was only marginally affected by the statistics used or by test error, estimated as 3.92%. The isolates were assigned to five multi-membered and 28 single-membered clusters defined by the simple matching coefficient at the 89% similarity level. With few exceptions, the isolates were sharply separated from theGordona marker strains. Essentially the same classification was obtained when the test strains were examined using a Curie-point pyrolysis mass spectrometric procedure. It can be concluded that the gordonae form a heterogeneous taxonomic group, the members of which can be distinguished from representatives of validly described species ofGordona.

We collected urban soil samples impacted by polycyclic aromatic hydrocarbons (PAHs) from a sorbent-based remediation field trial to address concerns about unwanted side-effects of 2% powdered (PAC) or granular (GAC) activated carbon amendment on soil microbiology and pollutant biodegradation. After three years, total microbial cell counts and respiration rates were highest in the GAC amended soil. The predominant bacterial community structure derived from denaturing gradient gel electrophoresis (DGGE) shifted more strongly with time than in response to AC amendment. DGGE band sequencing revealed the presence of taxa with closest affiliations either to known PAH degraders, e.g. Rhodococcus jostii RHA-1, or taxa known to harbor PAH degraders, e.g. Rhodococcus erythropolis, in all soils. Quantification by real-time polymerase chain reaction yielded similar dioxygenases gene copy numbers in unamended, PAC-, or GAC-amended soil. PAH availability assessments in batch tests showed the greatest difference of 75% with and without biocide addition for unamended soil, while the lowest PAH availability overall was measured in PAC-amended, live soil. We conclude that AC had no detrimental effects on soil microbiology, AC-amended soils retained the potential to biodegrade PAHs, but the removal of available pollutants by biodegradation was most notable in unamended soil.

Background In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs) present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. Results AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high frequencies. Conclusions AmpliconNoise followed by Perseus is a very effective pipeline for the removal of noise. In addition the principles behind the algorithms, the inference of true sequences using Expectation-Maximization (EM), and the treatment of chimera detection as a classification or 'supervised learning' problem, will be equally applicable to new sequencing technologies as they appear.

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.

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10 3 to 10 4 and 10 4 to 10 5 bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.

Autotrophic ammonia-oxidizing bacteria (AOB) are of vital importance to wastewater treatment plants (WWTP), as well as being an intriguing group of microorganisms in their own right. To date, corroboration of quantitative measurements of AOB by fluorescence in situ hybridization (FISH) has relied on assessment of the ammonia oxidation rate per cell, relative to published values for cultured AOB. Validation of cell counts on the basis of substrate transformation rates is problematic, however, because published cell-specific ammonia oxidation rates vary by over two orders of magnitude. We present a method that uses FISH in conjunction with confocal scanning laser microscopy to quantify AOB in WWTP, where AOB are typically observed as microcolonies. The method is comparatively simple, requiring neither detailed cell counts or image analysis, and yet it can give estimates of either cell numbers or biomass. Microcolony volume and diameter were found to have a log-normal distribution. We were able to show that virtually all (>96%) of the AOB biomass occurred as microcolonies. Counts of microcolony abundance and measurement of their diameter coupled with a calibration of microcolony dimensions against cell numbers or AOB biomass were used to determine AOB cell numbers and biomass in WWTP. Cell-specific ammonia oxidation rates varied between plants by over three orders of magnitude, suggesting that cell-specific ammonia oxidation is an important process variable. Moreover, when measured AOB biomass was compared with process-based estimates of AOB biomass, the two values were in agreement. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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.