Potential of predominant activated sludge bacteria as recipients in conjugative plasmid transfer

We investigated the possibility of conjugative plasmid transfer to the predominant bacteria in activated sludge and the factors influencing the transfer frequency in the activated sludge process. We performed conjugative transfers of a self-transmissible, broad-host-range plasmid RP4 from Escherichia coli C600 to activated sludge bacteria by broth mating. Most of the activated sludge bacteria tested could acquire plasmid RP4, although the transfer frequencies varied from 8.8 x 10(-7) to 1.3 x 10(-2) transconjugants per recipient. The transfer frequencies in several strains were similar to, or higher than, that in intraspecific transfer to E. coli HB101. Matings under various environmental conditions showed that factors relevant to physiological activity, such as temperature and nutrient conditions, seemed to affect the transfer frequency. In addition, conjugative transfer was detected even in filtered raw and treated wastewaters. Thus, the predominant activated sludge bacteria seem to have sufficient potential as recipients in conjugative plasmid transfer under the conditions likely to occur in the activated sludge process. Transfer frequency was reduced by agitation in the presence of suspended solid. This may suggest that conjugative plasmid transfer is physically inhibited in aeration tanks.     

17beta-estradiol-degrading bacteria isolated from activated sludge

Fourteen phylogenetically diverse 17beta-estradiol-degrading bacteria (strains KC1-14) were isolated from activated sludge of a wastewater treatment plant. These isolates widely distributed among eight different genera--Aminobacter (strains KC6 and KC7), Brevundimonas (strain KC12), Escherichia (strain KC13), Flavobacterium (strain KC1), Microbacterium (strain KC5), Nocardioides (strain KC3), Rhodococcus (strain KC4), and Sphingomonas (strains KC8-KC11 and KC14)--of three Phyla: Proteobacteria, Actinobacteria, and Bacteroidetes. All 14 isolates were capable of converting 17beta-estradiol to estrone, but only three strains (strains KC6, KC7, and KC8) showed the ability to degrade estrone. Only strain KC8 could use 17beta-estradiol as a sole carbon source. Based on the degree of estrogens being transformed and the estrogenicity of metabolites and/ or end products of estrogen degradation, three different degradation patterns (patterns A-C) were observed from degradation tests using resting cells. Eleven out of 14 isolates showed degradation pattern A, where 17beta-estradiol was stoichiometrically converted to estrone. Estrone was confirmed to be a degradation product of 17beta-estradiol; however, estrone was not further degraded during the course of experiments. Strains KC6 and KC7 exhibited degradation pattern B, where both 17beta-estradiol and estrone were degraded, with slower 17beta-estradiol degradation rates than those observed in pattern A. Strain KC8 was the only strain exhibited degradation pattern C, where 17beta-estradiol and estrone were rapidly degraded within 3 days. No residual 17beta-estradiol and estrone or estrogenic activity was detected after 5 days, suggesting that strain KC8 could degrade 17beta-estradiol into nonestrogenic metabolites/end products. Strains KC6-8 exhibited nonspecific monooxygenase activity but not nonspecific dioxygenase activity. However, the relationship between nonspecific monooxygenase activity and its estrogen degradation ability was unclear.     

Isolation, characterization and identification of polyhydroxyalkanoate-accumulating bacteria from activated sludge

Two novel gram-positive bacteria capable of accumulating poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] were isolated from an anaerobic-oxic activated sludge system fed with acetate. Strains Lpha5 and Lpha7 are motile cocci, 1-2 microm in diameter, occurring singly or in pairs. These isolates have doubling times ranging from 0.4-1.7 d and can accumulate in high levels of poly(3HB-co-3HV) (up to 44.7% of cell dry weight) when grown on complex media. Furthermore, these two strains exhibited the rapid substrate uptake and accumulation of storage granules as observed in situ. Under aerobic conditions, about 14.4% (cell dry weight) polyhydroxyalkanoate and 82% (carbon dry weight) cellular carbohydrate were produced from acetate and glucose, respectively. Under anaerobic conditions, poly(3HB-co-3HV) and cellular carbohydrate accumulated when glucose but not acetate was fed. The result of analysis of 16S rRNA sequence revealed that both strains belong to the gram-positive high-G + C group, but are significantly different from their closest phylogenetic relatives, Dermatophilus sp. and Terrabacter sp., to warrant classification as a new species.     

Oxygen tolerance of sulfate-reducing bacteria in activated sludge

The oxygen tolerance of sulfate-reducing bacteria (SRB) present in activated sludge was studied in batch incubations using radiolabeled [35S]sulfate and a most probable number (MPN) technique employing activated sludge medium. Sulfate reduction (SR) could not be detected in activated sludge during oxic incubation or in the presence of nitrate. However, upon anoxic incubation of both freshly sampled activated sludge and activated sludge preaerated for 40 min, SR resumed immediately at an initial rate of 2 microM h(-1). During long-term aeration of activated sludge, the number of viable and culturable SRB remained constant at around 10(6) SRB mL(-1) throughout a 121 h aeration period. During the first 9 h of the 121 h aeration period, the anaerobic SR activitywas unaffected, as compared to that of an unaerated control sample, and recommenced instantaneously upon anoxic incubation. Even after 121 h of continuous aeration, SR took place within 1.5 h after anoxic incubation albeit at a rate less than 20% that of the unaerated control. As suggested by MPN estimates and the observed kinetics of SR, oxygen exposure resulted in temporary metabolic inactivation of SRB but did not cause cell death. Consequently, SRB have the potential for quick proliferation during anoxic storage of activated sludge.     

Production of anti-Gordonia amarae mycolic acid polyclonal antibody for detection of mycolic acid-containing bacteria in activated sludge foam

Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti-Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 microg. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti-G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge.     

大肠杆菌L-阿拉伯糖异构酶的克隆表达及D-塔格糖的制备

将大肠杆菌K-12中的L-阿拉伯糖异构酶基因araA克隆到载体pET-28a上,并转化入大肠杆菌BL21(DE3)中表达.通过SDS-PAGE分析发现,重组菌株能表达出大量可溶性酶蛋白.以重悬菌液为酶源、D-半乳糖为底物,对酶转化D-塔格糖的条件进行测定.结果表明:D-塔格糖的最佳转化温度为60℃.在pH79的范围中,D-塔格糖的转化率均能达到30%40%.加入Mn2+、Ca2+、Co2+和Mg2+均能够使D-塔格糖的转化率提高,EDTA处理后的酶明显不具备催化能力,加入Mn2+后能使酶液恢复催化能力,但并不随着Mn2+浓度的提高而增大.     

Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine

Susceptibility to 12 antibiotics was tested in 75 unrelated lactic acid bacteria strains of wine origin of the following species: 38 Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 Oenococcus oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici, and 3 Leuconostoc mesenteroides. The Minimal Inhibitory Concentrations of the different antibiotics that inhibited 50% of the strains of the Lactobacillus, Leuconostoc and Pediococcus genera were, respectively, the following ones: penicillin (2, < or =0.5, and < or =0.5 microg/ml), erythromycin (< or =0.5 microg/ml), chloramphenicol (4 microg/ml), ciprofloxacin (64, 8, and 128 microg/ml), vancomycin (> or =128 microg/ml), tetracycline (8, 2, and 8 microg/ml), streptomycin (256, 32, and 512 microg/ml), gentamicin (64, 4, and 128 microg/ml), kanamycin (256, 64, and 512 microg/ml), sulfamethoxazole (> or =1024 microg/ml), and trimethoprim (16 microg/ml). All 21 O. oeni showed susceptibility to erythromycin, tetracycline, rifampicin and chloramphenicol, and exhibited resistance to aminoglycosides, vancomycin, sulfamethoxazole and trimethoprim, that could represent intrinsic resistance. Differences were observed among the O. oeni strains with respect to penicillin or ciprofloxacin susceptibility. Antibiotic resistance genes were studied by PCR and sequencing, and the following genes were detected: erm(B) (one P. acidilactici), tet(M) (one L. plantarum), tet(L) (one P. parvulus), aac(6')-aph(2") (four L. plantarum, one P. parvulus, one P. pentosaceus and two O. oeni), ant(6) (one L. plantarum, and two P. parvulus), and aph(3')-IIIa (one L. plantarum and one O. oeni). This is the first time, to our knowledge, that ant(6), aph(3')-IIIa and tet(L) genes are found in Lactobacillus and Pediococcus strains and antimicrobial resistance genes are reported in O. oeni strains.     

Treatment of wastewater from a whey processing plant using activated sludge and anaerobic processes

Wastewater from a whey processing plant was treated in two on-site pilot plants, a three-stage activated sludge plant and an anaerobic reactor, each of which had the capacity of treating 230 L/h of wastewater. The activated sludge treatment was very effective. It reduced 99% of 5-d biochemical oxygen demand of the plant wastewater (from an average of 1062 to 9 mg/L) and 91% of total Kjeldahl nitrogen (from 109 to 10 mg/L) after a total retention time of 19.8 h. The intermediate 5-d biochemical oxygen demand reductions were 86% after 3.8 h in the first stage and 97% after another 8 h in the second stage. The completely mixed anaerobic reactor reduced only 87% of 5-d biochemical oxygen demand after 2 d of retention. However, with an additional 8 h of activated sludge treatment the total 5-d biochemical oxygen demand was reduced by 99%. Both pilot plants were operated smoothly in spite of the considerable fluctuations in pollutant levels of the plant wastewater.     

Acute and chronic responses of activated sludge viability and performance to silica nanoparticles

Recently, the potential health and environmental risks of silica nanoparticles (SiO(2) NPs) are attracting great interest. However, little is known about their possible impacts on wastewater biological nitrogen and phosphorus removal. In this study, the acute and chronic effects of SiO(2) NPs on activated sludge viability and biological nutrient removal performance were investigated. It was found that the presence of environmentally relevant concentration (1 mg/L) of SiO(2) NPs caused no adverse acute and chronic effects on sludge viability and wastewater nitrogen and phosphorus removal. However, chronic exposure to 50 mg/L SiO(2) NPs induced the increase of effluent nitrate concentration, and thus depressed the total nitrogen (TN) removal efficiency from 79.6% to 51.6% after 70 days of exposure, which was due to the declined activities of denitrifying enzymes, nitrate reductase and nitrite reductase. Wastewater phosphorus removal was insensitive to 1 and 50 mg/L SiO(2) NPs after either the acute or chronic exposure, because the critical factors closely related to biological phosphorus removal were not significantly changed, such as the activities of exopolyphosphatase and polyphosphate kinase and the intracellular transformations of polyhydroxyalkanoates and glycogen. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that the bacterial community structure was changed after long-term exposure to 50 mg/L SiO(2) NPs, and the quantitative PCR assays indicated that the abundance of denitrifying bacteria was decreased, which was consistent with the declined wastewater nitrogen removal.     

Investigation of sorption behavior between pyrene and colloidal organic carbon from activated sludge processes

The sorption behavior of pyrene for different size fractions of colloidal organic carbon (COC) originating from two biological wastewater treatment facilities (a full-scale activated sludge system (FSAS) and a membrane bioreactor (MBR)) was investigated by fluorescence quenching. Fluorescence lifetime measurements demonstrated a dynamic quenching component in all samples, including the colloidal-free filtrates. COC sorption coefficients (Kcoc) for pyrene ranged from <1 x 10(3) L/kgcoc to 80 x 10(3) L/kgcoc and were comparable to values obtained in the literature for natural organic matter. Both linear and nonlinear behaviors were observed in the Freundlich-described converted isotherms (n = 0.89-2.1). The aromatic content of COC was quantified by the molar extinction coefficient at 280 nm (e280). Good correlations were observed between COC-pyrene sorption coefficients and e280 coefficients, suggesting that e280 may be a useful tool for predicting colloidal transport of hydrophobic organic compounds (HOCs) from activated sludge systems. The removal of COC from treated effluents may appreciably reduce the concentrations of HOCs discharged to receiving streams.     

Diagnosis and quantification of glycerol assimilating denitrifying bacteria in an integrated fixed-film activated sludge reactor via 13C DNA stable-isotope probing

Glycerol, a byproduct of biodiesel and oleo-chemical manufacturing operations, represents an attractive alternate to methanol as a carbon and electron donor for enhanced denitrification. However, unlike methanol, little is known about the diversity and activity of glycerol assimilating bacteria in activated sludge. In this study, the microbial ecology of glycerol assimilating denitrifying bacteria in a sequencing batch integrated fixed film activated sludge (SB-IFAS) reactor was investigated using (13)C-DNA stable isotope probing (SIP). During steady state SB-IFAS reactor operation, near complete nitrate removal (92.7 ± 5.8%) was achieved. Based on (13)C DNA clone libraries obtained after 360 days of SB-IFAS reactor operation, bacteria related to Comamonas spp. and Diaphorobacter spp. dominated in the suspended phase communities. (13)C assimilating members in the biofilm community were phylogenetically more diverse and were related to Comamonas spp., Bradyrhizobium spp., and Tessaracoccus spp. Possibly owing to greater substrate availability in the suspended phase, the glycerol-assimilating denitrifying populations (quantified by real-time PCR) were more abundant therein than in the biofilm phase. The biomass in the suspended phase also had a higher specific denitrification rate than the biofilm phase (p = 4.33e-4), and contributed to 69.7 ± 4.5% of the overall N-removal on a mass basis. The kinetics of glycerol based denitrification by suspended phase biomass were approximately 3 times higher than with methanol. Previously identified methanol assimilating denitrifying bacteria were not associated with glycerol assimilation, thereby suggesting limited cross-utilization of these two substrates for denitrification in the system tested.     

Nano-TiO2 enhanced photofermentative hydrogen produced from the dark fermentation liquid of waste activated sludge

After anaerobic dark fermentation of waste activated sludge (WAS) for hydrogen production, there are a large number of organic compounds including protein, polysaccharide, and volatile fatty acids left in the dark fermentation liquid, which can be further bioconverted to hydrogen by photofermentation techniquea. In this study, the enhancement of photofermentative hydrogen produced from WAS dark fermentation liquid by using nano-TiO2 is reported. First, high concentration of NH(4)(+)-N in the dark fermentation liquid was observed to inhibit the photofermentative hydrogen production, and its removal was essential. Then the effect of nano-TiO2 on photofermentative hydrogen generation was investigated, and the addition of 100 mg/L nano-TiO2 increased hydrogen by 46.1%. Finally, the mechanisms for nano-TiO2 improving hydrogen production were investigated. It was found that nano-TiO2 improved the decomposition of protein and polysaccharide to small-molecule organic compounds and promoted the growth of photosynthetic bacteria and the activity of nitrogenase but decreased the H2-uptake hydrogenase activity.     

Elimination of selected acidic pharmaceuticals from municipal wastewater by an activated sludge system and membrane bioreactors

The elimination of six acidic pharmaceuticals (clofibric acid, diclofenac, ibuprofen, ketoprofen, mefenamic acid, and naproxen) in a real wastewater treatment plant (WWTP) using an activated sludge system and membrane bioreactors (MBRs) was investigated by using a gas chromatography/mass spectrometry (GC/MS) system for measurement of the compounds. Limited information is available for some of the tested pharmaceuticals at present. Solid retention times (SRTs) of the WWTP and the two MBRs were 7, 15, and 65 days, respectively. The elimination rates varied from compound to compound. The MBRs exhibited greater elimination rates for the examined pharmaceuticals than did the real plant. Dependency of the elimination rates of the pharmaceuticals on SRTs was obvious; the MBR operated with a longer SRT of 65 days clearly showed better performance than did the MBR with a shorter SRT of 15 days. The difference between the two MBRs was particularly significant in terms of elimination of ketoprofen and diclofenac. Measurements of the amounts of adsorbed pharmaceuticals on the sludge and aerobic batch elimination experiments were carried out to investigate the elimination pathways of the pharmaceuticals. Results of the batch elimination tests revealed that the sludges in the MBRs had large specific sorption capacities mainly due to their large specific surface areas. Despite the sorption capacities of sludges, the main mechanism of elimination of the pharmaceuticals in the investigated processes was found to be biodegradation. Biodegradation of diclofenac, which has been believed to be refractory to biodegradation, seemed to occur very slowly.     

A laboratory-scale test of anaerobic digestion and methane production after phosphorus recovery from waste activated sludge

In enhanced biological phosphorus removal (EBPR) processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP) intracellularly. We previously discovered that nearly all of polyP could be released from waste activated sludge simply by heating it at 70 degrees C for about 1 h. We also demonstrated that this simple method was applicable to phosphorus (P) recovery from waste activated sludge in a pilot plant-scale EBPR process. In the present study, we evaluated the effect of this sludge processing (heat treatment followed by calcium phosphate precipitation) on anaerobic digestion in laboratory-scale experiments. The results suggested that the sludge processing for P recovery could improve digestive efficiency and methane productivity at both mesophilic (37 degrees C) and thermophilic (53 degrees C) temperatures. In addition, heat-treated waste sludge released far less P into the digested sludge liquor than did untreated waste sludge. It is likely that the P recovery step prior to anaerobic digestion has a potential advantage for controlling struvite (magnesium ammonium phosphate) deposit problems in sludge handling processes.     

Transfer of Escherichia coli O157:H7 to romaine lettuce due to contact water from melting ice

Ice can be used to chill romaine lettuce and maintain relative humidity during transportation. Escherichia coli O157:H7 may contaminate water used for ice. The objective of this study was to determine the potential for E. coli O157:H7 contamination of romaine lettuce from either ice contaminated with the pathogen or by transfer from lettuce surfaces via melting ice. In experiment 1, lettuce was spot inoculated with E. coli O157:H7 and chilled with ice prepared from uncontaminated tap water. In experiment 2, water inoculated with this pathogen was frozen and used to ice lettuce. Three heads of lettuce were stacked in each container and stored at 4 or 20 degrees C. After the ice melted, E. coli O157:H7 attachment to and recovery from the lettuce leaves were determined. For experiment 1, the population of E. coli O157:H7 attached to inoculated sites averaged 3.8 and 5.5 CFU/cm2 at 4 and 20 degrees C, respectively. Most of the uninoculated sites became contaminated with the pathogen due to ice melt. For experiment 2, 3.5 to 3.8 log CFU E. coli O157:H7 per cm2 was attached to the top leaf on the first head. After rinsing with chlorinated water (200 microg/ml), E. coli O157:H7 remained on the surface of the top head (1.8 to 2.0 log CFU/cm2). There was no difference in numbers of E. coli O157:H7 recovered from each sampling site at 4 and 20 degrees C. Results show that E. coli O157:H7 can be transferred onto other produce layers in shipping containers from melted ice made of contaminated water and from contaminated to uncontaminated leaf surfaces.     

Incidence and antibiotic susceptibility of bacteriocin‐producing lactic acid bacteria from dairy products

Screening for bacteriocin production by strains of lactic acid bacteria (LAB) from local dairy products in Iran resulted in the detection of 10 bacteriocin‐producing strains. Among 105 isolated, 10 bacteriocin producers were phenotypically and genotypically identified as Enterococcus spp. The antimicrobial compounds produced by these novel strains were inactivated by trypsin, proteinase k. These bacteriocins also were active in a wide range of pH and temperature values, and inhibited not only the closely related LAB, but also Listeria monocytogenes.     

混合菌同步糖化共发酵造纸污泥产乙醇

对酿酒酵母（Saccharomyces cerevisiae）与重组大肠杆菌K011（Escherichia coli）混合菌同步糖化共发酵造纸污泥产乙醇进行了初步研究。在底物浓度为50g／L时,通过单因素实验和正交实验获得乙醇发酵的最佳条件：纤维素酶添加量25FPU／g底物,接种量为6％,酿酒酵母与重组大肠杆菌K011接种比例为1：1（细胞干重初始浓度分别为1．0g／L和0．3g／L左右）。发酵72h后,乙醇浓度为5．71g／L,产率达到0．114g乙醇／g污泥,达到理论值的42．5％。分别用酿酒酵母、K011单菌种发酵与双菌株组合发酵对比结果表明,混合菌发酵效果明显优于单菌种发酵。