Ensuring safe reuse of residential wastewater: reduction of microbes and genes using peat biofilter and batch chlorination in an on-site treatment system.

AIMS: A batch chlorination system was optimized for on-site wastewater treatment and reuse system (OWRS) and its efficiency was evaluated for reducing viruses, protozoa, bacteria and antimicrobial resistance in cold and warm seasons. METHODS AND RESULTS: The OWRS performance in reducing microbial contaminants was determined by assessing three different faecal indicators (Escherichia coli, F-specific coliphages and Clostridium perfringens for measuring the disinfection efficiency of bacteria, viruses and spore-formers and surrogate for protozoa, respectively) using culture-based methods. Quantitative PCR was used to quantify pathogenic bacteria (Shiga-toxin-producing E. coli (STEC), Campylobacter spp., and Arcobacter spp.), a human-associated faecal marker (gyrB), and tetracycline resistant bacteria (tetQ). The levels of E. coli, coliphages and Cl. perfringens showed 5·4, 2·3, 2·5 log reduction, respectively, upon disinfection. In the final effluents, coliphages (1·7 × 102 PFU 100 ml-1 ) and Cl. perfringens (3·4 CFU 100 ml-1 ) were detected in 80 and 100% of the samples, but E. coli was not found. The removal and inactivation of E. coli and Cl. perfringens were not significantly different across the seasons, however, efficacy of removal and inactivation of F-specific coliphage was significantly reduced during the winter/spring season compared to the summer/autumn season (P = 0·009). The reduction of Arcobacter, gyrB and tetQ by 3·1, 2·3 and 2·3 log, respectively, was mostly due to peat biofiltration under the study conditions. CONCLUSIONS: This study demonstrated that peat biofiltration was the most important step of the OWRS to remove microbes and genes from wastewater before spray irrigation of the effluents. The irrigation system is not suitable for edible crops because of the potential presence of residual pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: On-site wastewater treatment systems are a practical option for reusing the wastewater for landscape, especially for those areas where geological and seasonal limitations impact the removal of microbial contaminants by soil infiltration.

Screening of human enteric microorganisms for potential biotransformation of polycyclic aromatic hydrocarbons.

This study examined the potential for metabolism of select polycyclic aromatic hydrocarbons (PAH) by human enteric microorganisms. Experiments were performed under anaerobic conditions with various combinations of enteric microbial suspensions, PAH concentrations, nutrient mixtures, and time courses. No PAH metabolites were detected upon GC-TOF-MS analysis of samples digested by tetramethylammonium hydroxide thermochemolysis. No mineralization of (14)C-labeled phenanthrene was observed. These results suggest the lack of partial or complete metabolism of PAHs by enteric microorganisms and therefore the absence of major bioactivation pathways that would expose intestinal lining to potentially carcinogenic PAH metabolites.

Growth of sulfate-reducing bacteria with solid-phase electron acceptors.

Hannebachite (CaSO3 x 0.5H2O), gypsum (CaSO4 x 2H2O), anglesite (PbSO4), and barite (BaSO4) were tested as electron acceptors for sulfate-reducing bacteria with lactate as the electron donor. Hannebachite and gypsum are commonly associated with flue gas desulfurization products, and anglesite is a weathering product found in lead mines. Barite was included as the most insoluble sulfate. Growth of sulfate-reducing bacteria was monitored by protein and sulfide (dissolved H2S and HS-) measurements. Biogenic sulfide formation occurred with all four solid phases, and protein data confirmed that bacteria grew under these electron acceptor conditions. Sulfide formation from gypsum was almost comparable in rate and quantity to that produced from soluble sulfate salt (Na2SO4); hannebachite reduction to sulfide was not as fast. Anglesite as the electron acceptor was also reduced to sulfide in the solution phase and galena (PbS) was detected in solids retrieved from spent cultures. Barite as the electron acceptor supported the least amount of growth and H2S formation. The results demonstrate that low-solubility crystalline phases can be biologically reactive under reducing conditions. Furthermore, the results demonstrate that galena precipitation through sulfide production by sulfate-reducing bacteria serves as a lead enrichment mechanism, thereby also alleviating the potential toxicity of lead. In view of the role of acidophilic thiobacilli in the oxidation of sulfides, the present work accentuates the role of anaerobic and aerobic microbes in the biogeochemical cycling of solid-phase sulfates and sulfides.

Characterization of chemotactic responses and flagella of Hyphomicrobium strain W1-1B.

Motile swarmer cells of Hyphomicrobium strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff's staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of Rhizobium meliloti cross-reacted with the striated flagella of Hyphomicrobium strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope regions.

Biodegradation of the acetanilide herbicides alachlor, metolachlor, and propachlor.

Alachlor, metolachlor, and propachlor are detoxified in biological systems by the formation of glutathione-acetanilide conjugates. This conjugation is mediated by glutathione-S-transferase, which is present in microorganisms, plants, and mammals. Other organic sulfides and inorganic sulfide also react through a nucleophilic attack on the 2-chloro group of acetanilide herbicides, but the products are only partially characterized. Sorption in soils and sediments is an important factor controlling the migration and bioavailability of these herbicides, while microbial degradation is the most important factor in determining their overall fate in the environment. The biodegradation of alachlor and metolachlor is proposed to be only partial and primarily cometabolic, and the ring cleavage seems to be slow or insignificant. Propachlor biodegradation has been reported to proceed to substantial (> 50%) mineralization of the ring structure. Reductive dechlorination may be one of the initial breakdown mechanisms under anaerobic conditions. Aerobic and anaerobic transformation products vary in their polarity and therefore in soil binding coefficient. A catabolic pathway for chloroacetanilide herbicides has not been presented in the literature because of the lack of mineralization data under defined cultural conditions.

Phylogenetic and narG analysis of a Hyphomicrobium isolate.

An obligately methylotrophic organism was isolated from a water well that manifested symptoms of biofouling. The isolate was appendaged and utilized methylamine, dimethylamine, trimethylamine, or methanol as the sole carbon and energy source. The isolate exhibited hydroxypyruvate reductase activity, suggesting C1-assimilation via the serine pathway. Fatty acid profiling indicated the predominance of 18:1 cis-fatty acids. The isolate did not grow anaerobically with nitrate as the final electron acceptor. Genomic DNA from the isolate did not hybridize against the narG gene, which encodes the alpha subunit of dissimilatory nitrate reductase in Escherichia coli. The phenotypic data suggested the assignment of the isolate to the genus Hyphomicrobium. The identification was supported by phylogenetic characterization based on 16S rRNA sequence comparisons of the isolate.

Insertion sequence IST3091 of Thiobacillus ferrooxidans.

An insertion sequence, designated as IST3091, was located adjacent to the putative origin of replication region of plasmid pTFI91 of Thiobacillus ferrooxidans TFI-91. The DNA sequence of the transposase gene of IST3091 revealed similarity with that of IS30, IS1086, IS4351, and the integrase gene of SpV1-R8A2 B (a bacteriophage of Spiroplasma citri). The sequence of IST3091 is 1063 bp long with partially matched 30-bp terminal inverted repeats. Several restriction fragments of plasmid pTFI91 of T. ferrooxidans containing the IST3091 element were cloned into the vector pHSG398. The hybrid plasmids (pBTL) were transformed into Escherichia coli NK7379 containing a miniF plasmid, which was devoid of transposable elements. The transposition function of the IST3091 element was confirmed by mobilizing hybrid plasmids via conjugation from transformed E. coli NK7379 (donor) to E. coli M8820 (recipient). The presence of the transposed element in transconjugants was detected by polymerase chain reaction amplification.

Biodegradation of atrazine in surface soils and subsurface sediments collected from an agricultural research farm.

The purpose of the present study was to assess atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) mineralization by indigenous microbial communities and to investigate constraints associated with atrazine biodegradation in environmental samples collected from surface soil and subsurface zones at an agricultural site in Ohio. Atrazine mineralization in soil and sediment samples was monitored as 14CO2 evolution in biometers which were amended with 14C-labeled atrazine. Variables of interest were the position of the label ([U-14C-ring]-atrazine and [2-14C-ethyl]-atrazine), incubation temperature (25 degrees C and 10 degrees C), inoculation with a previously characterized atrazine-mineralizing bacterial isolate (M91-3), and the effect of sterilization prior to inoculation. In uninoculated biometers, mineralization rate constants declined with increasing sample depth. First-order mineralization rate constants were somewhat lower for [2-14C-ethyl]-atrazine when compared to those of [U-14C-ring]-atrazine. Moreover, the total amount of 14CO2 released was less with [2-14C-ethyl]-atrazine. Mineralization at 10 degrees C was slow and linear. In inoculated biometers, less 14CO2 was released in [2-14C-ethyl]-atrazine experiments as compared with [U-14C-ring]-atrazine probably as a result of assimilatory incorporation of 14C into biomass. The mineralization rate constants (k) and overall extents of mineralization (Pmax) were higher in biometers that were not sterilized prior to inoculation, suggesting that the native microbial populations in the sediments were contributing to the overall release of 14CO2 from [U-14C-ring]-atrazine and [2-14C-ethyl]-atrazine. A positive correlation between k and aqueous phase atrazine concentrations (Ceq) in the biometers was observed at 25 degrees C, suggesting that sorption of atrazine influenced mineralization rates. The sorption effect on atrazine mineralization was greatly diminished at 10 degrees C. It was concluded that sorption can limit biodegradation rates of weakly-sorbing solutes at high solid-to-solution ratios and at ambient surface temperatures if an active degrading population is present. Under vadose zone and subsurface aquifer conditions, however, low temperatures and the lack of degrading organisms are likely to be primary factors limiting the biodegradation of atrazine.

Microbiological degradation of the herbicide dicamba.

Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba by P. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100-2000 mg dicamba L-1 (0.5-9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl- per mol of dicamba degraded. The cultures had an optimum pH 6.5-7.0 for dicamba degradation. Growth studies at 10 degrees C, 20 degrees C, and 30 degrees C yielded activation energy values in the range of 19-36 kcal mol-1 and an average of Q10 value of 4.0. Compared with the pure culture P. paucimobilis, the consortium was more active at the lower temperature.

Characterization of the pTFI91-family replicon of Thiobacillus ferrooxidans plasmids.

Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5 alpha cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.

Degradation and mineralization of atrazine by a soil bacterial isolate.

An atrazine-degrading bacterial culture was isolated from an agricultural soil previously impacted by herbicide spills. The organism was capable of using atrazine under aerobic conditions as the sole source of C and N. Cyanuric acid could replace atrazine as the sole source of N, indicating that the organism was capable of ring cleavage. Ring cleavage was confirmed in 14CO2 evolution experiments with [U-14C-ring]atrazine. Between 40 and 50% of ring-14C was mineralized to 14CO2. [14C]biuret and [14C]urea were detected in spent culture media. Cellular assimilation of 14C was negligible, in keeping with the fully oxidized valence of the ring carbon. Chloride release was stoichiometric. The formation of ammonium during atrazine degradation was below the stoichiometric amount, suggesting a deficit due to cellular assimilation and metabolite-N accumulation. With excess glucose and with atrazine as the sole N source, free ammonium was not detected, suggesting assimilation into biomass. The organism degraded atrazine anaerobically in media which contained (i) atrazine only, (ii) atrazine and glucose, and (iii) atrazine, glucose, and nitrate. To date, this is the first report of a pure bacterial isolate with the ability to cleave the s-triazine ring structure of atrazine. It was also concluded that this bacterium was capable of dealkylation, dechlorination, and deamination in addition to ring cleavage.

Oxidative dissolution of arsenopyrite by mesophilic and moderately thermophilic acidophiles.

The purpose of this work was to determine solution- and solid-phase changes associated with the oxidative leaching of arsenopyrite (FeAsS) by Thiobacillus ferrooxidans and a moderately thermoacidophilic mixed culture. Jarosite [KFe(3)(SO(4))(2)(OH)(6)], elemental sulfur (S), and amorphous ferric arsenate were detected by X-ray diffraction as solid-phase products. The oxidation was not a strongly acid-producing reaction and was accompanied by a relatively low redox level. The X-ray diffraction lines of jarosite increased considerably when ferrous sulfate was used as an additional substrate for T. ferroxidans. A moderately thermoacidophilic mixed culture oxidized arsenopyrite faster at 45 degrees C than did T. ferroxidans at 22 degrees C, and the oxidation was accompanied by a nearly stoichiometric release of Fe and As. The redox potential was initially low but subsequently increased during arsenopyrite oxidation by the thermoacidophiles. Jarosite, S, and amorphous ferric arsenate were also formed under these conditions.

Maximum temperature limits for acidophilic, mesophilic bacteria in biological leaching systems.

The maximum temperature for growth (T(max)) was determined for pure and mixed cultures of acidophilic thiobacilli. The experimental system was based on incubating the cultures in liquid media exposed to a linear temperature gradient. The T(max) values varied within the range of 36.1 to 43.6 degrees C.

Nutrient effect on the biological leaching of a black-schist ore.

The purpose of the study was to examine the influence of inorganic N (NH(4), NO(3)) and phosphate on the biological oxidation of a sulfidic black-schist ore which contained pyrrhotite as the main iron sulfide. Iron was initially solubilized as Fe from the ore and subsequently oxidized to Fe in shake flask experiments. Under these experimental conditions, iron dissolution from pyrrhotite was mainly a chemical reaction, with some enhancement by bacteria, whereas the subsequent Fe oxidation was bacterially mediated, with negligible contribution from chemical oxidation. Phosphate amendment did not enhance Fe oxidation. Chemical analysis of leach solutions with no exogenous phosphate revealed that phosphate was solubilized from the black-schist ore. Ammonium amendment (6 mM) enhanced Fe oxidation, whereas the addition of nitrate (6 and 12 mM) had a negative effect. An increase in the temperature from 30 to 35 degrees C slightly enhanced Fe oxidation, but the effect was statistically not significant. The precipitation of potassium jarosite was indicative of Fe oxidation and was absent in nitrate-inhibited cultures because of the lack of Fe oxidation. The black-schist ore also contained phlogopite, which was altered to vermiculite in iron-oxidizing cultures.

Mineral Products of Pyrrhotite Oxidation by Thiobacillus ferrooxidans.

The biological leaching of pyrrhotite (Fe(1-x)S) by Thiobacillus ferrooxidans was studied to characterize the oxidation process and to identify the mineral weathering products. The process was biphasic in that an initial phase of acid consumption and decrease in redox potential was followed by an acid-producing phase and an increase in redox potential. Elemental S was one of the first products of pyrrhotite degradation detected by X-ray diffraction. Pyrrhotite oxidation also yielded K-jarosite [KFe(3)(SO(4))(2)(OH)(6)], goethite (alpha-FeOOH), and schwertmannite [Fe(8)O(8)(OH)(6)SO(4)] as solid-phase products. Pyrrhotite was mostly depleted after 14 days, whereas impurities in the form of pyrite (cubic FeS(2)) and marcasite (orthorhombic FeS(2)) accumulated in the leach residue.

Role of divalent cations in the subunit associations of complex flagella from Rhizobium meliloti.

Rhizobium meliloti, a symbiotic, nitrogen-fixing soil bacterium with complex flagella, as well as other members of the family Rhizobiaceae, rapidly lost motility when suspended in buffers lacking divalent cations but retained good motility in buffers containing calcium, magnesium, barium, or strontium. Loss of motility was associated with loss of flagella from the cells. Analysis of flagella by sedimentation, gel electrophoresis, and electron microscopy revealed that removal of divalent cations from the complex flagella of R. meliloti resulted in extensive dissociation of the flagellar filaments into low-molecular-weight subunits. Accordingly, divalent cations such as calcium and magnesium that are normally present at high concentrations in the soil solution may be crucial to the assembly and rigidity of complex flagella.

Solid-phase products of bacterial oxidation of arsenical pyrite.

Bacterial leaching of an As-containing pyrite concentrate produced acidic (pH < 1) leachates. During the leaching, the bacteria solubilized both As and Fe, and these two elements were distributed in solution-phase and solid-phase products. Jarosite and scorodite were the exclusive crystalline products in precipitate samples from the bacterial leaching of the sulfide concentrate.

Bacterial oxidation of sulfide minerals in column leaching experiments at suboptimal temperatures.

The purpose of the work was to quantitatively characterize temperature effects on the bacterial leaching of sulfide ore material containing several sulfide minerals. The leaching was tested at eight different temperatures in the range of 4 to 37 degrees C. The experimental technique was based on column leaching of a coarsely ground (particle diameter, 0.59 to 5 mm) ore sample. The experimental data were used for kinetic analysis of chalcopyrite, sphalerite, and pyrrhotite oxidation. Chalcopyrite yielded the highest (73 kJ/mol) and pyrrhotite yielded the lowest (25 kJ/mol) activation energies. Especially with pyrrhotite, diffusion contributed to rate limitation. Arrhenius plots were also linear for the reciprocals of lag periods and for increases of redox potentials (dmV/dt). Mass balance analysis based on total S in leach residue was in agreement with the highest rate of leaching at 37 and 28 degrees C. The presence of elemental S in leach residues was attributed to pyrrhotite oxidation.

Chemical inactivation of microorganisms on rigid gas permeable contact lenses.

The efficiency of four contact lens disinfectant formulations was tested against three types of bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, and Serratia marcescens) and one fungus (Aspergillus niger). The bacteria were tested both as free suspensions and after attachment on surfaces of rigid gas permeable contact lenses (RGPCL). The disinfection data were used to calculate the death rates and decimal reduction times for the test organisms. P. aeruginosa was the most sensitive and A. niger was the most resistant organism to the disinfectants. Scanning electron micrographs showed that P. aeruginosa occurred mostly as single cells, with little extracellular material, on the lens surface. In contrast, S. marcescens produced copious amounts of capsular material layered on the lens surface, promoting cell aggregation. Transmission electron micrographs revealed that bacterial cells were physically separated from the lens surface by a space barricaded with capsular material.