Evaluation of <Emphasis Type="Italic">smtA</Emphasis> expression and <Emphasis Type="Italic">E. coli</Emphasis> survival against cadmium ions

Cadmium and other heavy metals lead to environmental danger, and these heavy metals are a great threat to human and other animal’s health. Investigation of the relationship between survival of E. coli and metallothionein smtA gene expression against cadmium ion is the goal of this research. Survival of recombinant bacteria containing smtA gene was analyzed against various concentrations of cadmium chloride salt using optical density (OD). At the resistive range, recombinant bacteria were subjected to different treatments. At the logarithmic phase of bacterial growth, sampling, RNA extraction and cDNA synthesis were performed and smtA gene expression was then analyzed by real-time PCR using designed primers for smtA gene and Amp resistance (as the calibrator gene). Relative gene expression was calculated using the ??C_t method. The resistive range against cadmium chloride was 0.5–0.7 mM (minimum inhibitory concentration (MIC = 0.5 mM)). Survival and gene expression analysis showed that in induced bacteria, smtA expression was increased significantly that in turn conferred resistance to cadmium chloride prominently. There was a direct relationship between increased smtA gene expression and survival of the recombinant bacteria. Therefore, our result may help to confront to cadmium metal environmental pollution using overexpression of smtA gene expression in recombinant bacteria.     

Drinking water quality in Nepal’s Kathmandu Valley: a survey and assessment of selected controlling site characteristics

Water was sampled from over 100 sources in Nepal’s Kathmandu Valley, including municipal taps, dug wells, shallow-aquifer tube wells, deep-aquifer tube wells, and dhunge dharas (or stone spouts, public water sources that capture groundwater or surface water). Information was gathered on user preference and site and well characteristics, and water was examined for indicators of contamination from sewage, agriculture, or industry. Most problematic were total coliform and Escherichia coli bacteria, which were present in 94 and 72% of all the water samples, respectively. Contamination by nitrate, ammonia and heavy metals was more limited; nitrate and ammonia exceeded Nepali guidelines in 11 and 45% of the samples, respectively. Arsenic and mercury exceeded WHO guidelines in 7 and 10% of the samples, respectively, but arsenic never exceeded the less strict Nepali guideline. Significant differences existed in contamination levels between types of sources; dug wells and dhunge dharas, being the shallowest, were the most contaminated by bacteria and nitrate; deep-aquifer tube wells were the most contaminated by arsenic. Whereas E. coli concentrations decreased with depth, iron and ammonia concentrations increased with depth. These relationships account for people choosing to drink water with higher levels of bacterial contamination based on its superior (non-metallic) taste and appearance.     

Plasmid profile and curing analysis of Pseudomonas aeruginosa as metal resistant

The isolate Pseudomonas aeruginosa exhibited resistance to heavy metals such as cadmium, chromium, nickel and lead. Plasmid DNA was isolated from P. aeruginosa and designated as pBC15. The size of the plasmid DNA was approximately 23 kb. Escherichia coli DH5α was transformed with plasmid pBC15 subsequired resistance to nickel and ampicillin. The same size of the plasmid was isolated from E. coli transformant and separated on 0.7 % agarose gel electrophoresis. The restriction analysis of pBC15 showed that the plasmid DNA has single site for Bam HI and Eco RI and three sites for Xho I which were compared with 1 Kb DNA and λ Hind III digest molecular markers. Therefore, the size of the plasmid DNA of pBC15 was confirmed to be 23 kb. Curing was carried out by ethidium bromide, acridine orange, novobiocin, sodium dodechyl sulphate and elevated temperature (40 °C). Transformation and curing results suggest that nickel and ampicillin resistance gene was conferred by plasmid DNA. Cadmium resistant gene was present on chromosomal DNA along with the gene for chromium resistance. Lead resistance gene was shown to be present on the chromosomal DNA rather than the plasmid DNA as the cured and uncured cultures remained similar in lead resistance. Therefore, the ability of P. aeruginosa resistant to nickel and ampicillin is plasmid mediated and transferable to other strains whereas cadmium, chromium and lead could be chromosomal encoded. The heavy metal and antibiotic resistances of P. aeruginosa can be used to exploit for clean up industrial wastewater and bioremediation of heavy metal contaminated soil.     

Influence of montmorillonite on antimicrobial activity of tetracycline and ciprofloxacin

Antibiotics are used not only to fight infections and inhibit bacterial growth, but also as growth promotants in farm livestock. Farm runoff and other farm-linked waste have led to increased antibiotic levels present in the environment, the impact of which is not completely understood. Soil, more specifically clays, that the antibiotic contacts may alter its effectiveness against bacteria. In this study a swelling clay mineral montmorillonite was preloaded with antibiotics tetracycline and ciprofloxacin at varying concentrations and bioassays were conducted to examine whether the antibiotics still inhibited bacterial growth in the presence of montmorillonite. Escherichia coli was incubated with montmorillonite or antibiotic-adsorbed montmorillonite, and then the number of viable bacteria per mL was determined. The antimicrobial activity of tetracycline was affected in the presence of montmorillonite, as the growth of non-resistant bacteria was still found even when extremely high TC doses were used. Conversely, in the presence of montmorillonite, ciprofloxacin did inhibit E. coli bacterial growth at high concentrations. These results suggest that the effectiveness of antimicrobial agents in clayey soils depends on the amount of antibiotic substance present, and on the interactions between the antibiotic and the clays in the soil, as well.     

Risk of water pollution due to ash–sludge mixtures: column trials

The purpose of this work was to study the risks of water pollution due to the use of mixtures containing wood ash and sewage sludge. Mixtures including sludge and ash may be recycled as fertilizers, and they are economical as they do not integrate commercial limes, but Escherichia coli counts may keep significantly high in such mixtures, because their pH is not alkaline enough. In view of that, it seems interesting to study the E. coli survival in lixiviate from ash?Csludge mixtures including limes rather than from ash?Csludge mixtures alone. Two kinds of experiments were performed using laboratory column trials under saturated flow conditions. The first experiment investigated the chemical leaching behaviour of a mixture of 70% timber-industry wood ashes and 30% urban sewage sludge (% dry weight) at doses equivalent to 10 and 30?Mg/ha. The second experiment studied the survival of E. coli in lixiviates generated from 30?Mg/ha of a mixture consisting in 75% wood ash, 20% sewage sludge and 5% quicklime (% dry weight). In the first experiment, admixture of the ash and sludge achieved a stabilization of elements such as aluminium, iron, magnesium, nickle, carbon monoxide, cadmium, chromium and molybdenum that reduced their solubility compared with that in the ash or sludge alone. Significant solubilisation of heavy metals was not observed, with overall minor risk of chemical water pollution. In the second experiment, although including quicklime E. coli counts were still detected in the lixiviate, indicating risk of water contamination.     

Retention of strontium,cesium, lead and uranium by bacterial iron oxides from a subterranean environment

Bacteriogenic Fe oxides (BIOS) and groundwater samples were collected 195 m underground at the Stråssa Mine in central Sweden. Ferrous iron oxidizing bacteria, including stalked Gallionella ferruginea and filamenous Leptothrix sp., were prominent in the BIOS samples. The BIOS samples were found to contain only poorly ordered (amorphous) hydrous ferric oxide, as determined by X-ray diffraction. Inductively coupled plasma mass spectroscopy revealed hydroxylamine-reducible Fe and Mn oxide contents that ranged from 55 to 85% on a dry weight basis. Concentrations of Sr, Cs, Pb and U in filtered groundwater ranged from 0.002 to 1.8 μM. Solid phase concentrations of these heavy metals in the BIOS spanned the 0.04–2.23 mmol/kg range. Distribution coefficients (K_d values), calculated as the ratio between BIOS and dissolved heavy metal concentrations, revealed solid phase enrichments that, depending on the heavy metal and Fe oxide content of the sample, extended from 10^3.0 to 10^4.7. At the same time, however, a strong inverse linear relationship was found between log K_d values and the corresponding mass fraction of reducible oxide in the samples, implying that metal uptake was strongly influenced by the relative proportion of bacterial organic matter in the composite solids. Based on the metal accumulation properties of the BIOS, an important role can be inferred for intermixed Fe oxides and bacterial organic matter in the transport and fate of dissolved metals in groundwater systems.     

Bacterial consortium expressing surface displayed,intra- and extracellular lipases and pseudopyronine B for the degradation of oil

The lipA gene, encoding a solvent-tolerant extracellular lipase from Proteus sp. SW1, was displayed on the cell surface of Escherichia coli by fusing it to an antigen 43 anchoring motif. The display of LipA on the Escherichia coli cell surface was directly confirmed by immunofluorescence microscopy and flow cytometry. After 6 days of incubation in media containing 1 % used cooking oil, an Escherichia coli strain expressing surface displayed lipase was able to degrade 27 % of the oil. The biosurfactant, pseudopyronine B, was purified from culture supernatants of Pseudomonas sp. SL31. Its critical micelle concentration was determined to be 1400 mg/l, and the surfactant was stable within a temperature range from 0 to 120 °C and a pH range of 3–11. Pseudopyronine B-containing crude media extracts efficiently removed up to 51 % of the cadmium from contaminated water. We demonstrated the oil degradation ability of the mixed culture of four bacterial strains, namely the recombinant Escherichia coli expressing cell surface displayed lipase (pKKJlipA), His-tagged lipase (pETlipA), extracellular lipase-producing Proteus sp. SW1, and pseudopyronine B-producing Pseudomonas sp. SL31 by culturing in LB media containing 1 % oil. The consortium degraded 29 % of oil in one day and reached 84 % after 7 days.     

Biotransformation potential of hexavalent chromium by Bacillus pumilus-S4, Pseudomonas doudoroffii-S5 and Exiguobacterium-S8 in association with hydrophytes

Three chromium-resistant bacteria Bacillus pumilus-S4, Pseudomonas doudoroffii-S5 and Exiguobacterium-S8 were isolated from chromium-contaminated wastewater/soil and could resist very high concentrations of potassium chromate in Luria agar (up to 25 mg ml^?1) and acetate minimal medium (2 mg ml^?1). The strains showed growth at diverse pH and temperatures and could resist multiple heavy metals. Pseudomonas doudoroffii-S5 reduced (8.27 mg hexavalent chromium 24 h^?1) at a lower initial potassium chromate concentration (100 μg ml^?1), but overall more chromate (28.4 mg hexavalent chromium 24 h^?1) was reduced at a higher initial concentration (1,000 μg ml^?1). The addition of various heavy metals (zinc sulphate, copper sulphate, and manganese sulphate at 50 μg ml^?1) in the chromium reduction media did not significantly affect the hexavalent chromium reduction potential of these isolates. The chromium removal/detoxification potential of these strains increased when used in conjunction with hydrophytes Eichornia crassipes and Pistia stratiotes. Interestingly, the whole process runs automatically with less energy input, that is, the bacterial strains support the growth of plant while in turn the plant releases exudates that help bacterial growth.     

Assessment of heavy metal concentrations and associated resistant bacterial communities in bulk and rhizosphere soil of <Emphasis Type="Italic">Avicennia marina</Emphasis> of Pichavaram mangrove,India

Heavy metals are known to pose a potential threat to terrestrial and aquatic flora and fauna. Due to increasing human influence, heavy metal concentrations are rising in many mangrove ecosystems. Therefore, an assessment of heavy metal (Cd, Cr, Cu, Ni, Pb, Fe, Mn, and Zn) concentrations was conducted within the bulk soil and rhizosphere soil of Avicennia marina at the Pichavaram Mangrove Forest in India. The rhizosphere soil showed higher concentrations of metals than the bulk soil. Compared to the bulk soil, the metals Cd, Fe, Mn, and Zn were 6.0–16.7% higher, whereas Cr, Cu, Ni, and Pb were 1.7–2.8% higher concentration. Among the three selected sampling sites (dense mangrove forest, estuarine region, and sea region), the sea region had the highest concentration of all heavy metals except Zn. The trend of the mean metal concentration was Fe > Mn > Cr > Ni > Cu > Pb > Zn > Cd. Heavy metals concentrations elevated by the 2004 tsunami were persistent even after 4 years, due to sedimentary soil processes, the rhizosphere effect of mangroves, and anthropogenic deposition. Analysis of the heavy metal-resistant bacteria showed highest bacterial count for Cr-resistant bacteria and rhizosphere soil. The maximum level of heavy metal-resistant bacteria was observed at the site with the highest heavy metal contamination. The heavy metal-resistant bacteria can be used as indicator of heavy metal pollution and furthermore in bioremediation.     

EXAFS study on the cause of enrichment of heavy REEs on bacterial cell surfaces

Rare earth element (REE) pattern is a unique geochemical tracer and has been measured for various natural materials. Among these, the REE distribution pattern between bacteria and water exhibits anomalous enrichment in the heavy REE (HREE) part, which can act as a signature of bacteria-related materials in natural samples. In this study, the REE binding site on the cell surface of a Gram-positive bacterium (Bacillus subtilis) responsible for HREE enrichment has been identified using extended X-ray absorption fine structure (EXAFS) coupled with a study of the variation in REE distribution patterns. The EXAFS data showed that the HREEs form complexes with multiple phosphate site (including phosphoester site) with a larger coordination number (CN) at lower REE-bacteria ratios ([REE]/[bac]), while light and middle REEs form complexes to the phosphate site with a lower CN. The fraction coordinated to carboxylate increased for all REEs with increasing [REE]/[bac] ratio. On the other hand, the enrichment of HREE in the REE distribution patterns of the bacteria was less marked with increasing [REE]/[bac] ratio. This result is consistent with the EXAFS data, because the REE pattern of surface complex with multiple phosphate in a reference material exhibits a monotonous increase for heavier REE, while phosphate surface complex with a low CN and a carboxylate site reach a maximum around Sm and Eu. Based on these results, it is clear that the REE are primarily bound to the phosphate site and subsequently to the carboxylate site on the bacterial cell surface.Regarding the pH dependence in the range (3 < pH < 7), both the EXAFS and REE pattern data indicate that the fraction of REE-carboxylate increased as the pH increases. The results above obtained for B. subtilis were also valid for Escherichia coli, a Gram-negative bacterium, showing that similar phosphate and carboxylate sites are also available in the cell walls of E. coli, or other Gram negative bacteria. In all our results, the variation in REE patterns correlated with the binding site indicated by EXAFS, showing that the REE pattern itself reflects the binding site of the REE at the bacterial surface for various parameters (pH and [REE]/[bac] ratio). Thus, the REE patterns can be used to estimate the binding sites for lower [REE]/[bac] ratios where spectroscopic techniques cannot be applied.The average bond length between the REE and oxygen was compared for various REE sorbed on bacteria, showing that the bond length for HREE (Er to Lu) was much shorter than those extrapolated from the trend between La and Dy, because of the selective binding of the HREE as the multiple phosphate surface complexes. Our results are consistent with the selective enrichment of the HREE at the bacterial cell surfaces, considering that chemical species with a shorter bond length are more stable. Thus, it is clear that the HREE enrichment at the bacterial cell surfaces is caused by the formation of the multiple phosphate surface complexes. Based on these results, it is suggested that materials having such phosphate sites such as bacteria and bacteria-related materials can induce anomalous HREE enrichment in natural systems.     

An experimental model approach of biologically-assisted silicate dissolution with olivine and Escherichia coli – Impact on chemical weathering of mafic rocks and atmospheric CO2 drawdown

Chemical weathering of Mg, Ca-silicates and alumino-silicates contributes significantly to the drawdown of atmospheric CO₂ over long time scales. The present work focuses on how this mode of weathering may change in the presence of free-living bacteria in oligotrophic waters, which compose most of the surface freshwaters of the Earth. Forsterite (Fo90) was reacted for 1 week with a stable Escherichia coli population in water maintained at 37 °C and neutral pH in a batch reactor. Control samples with suspensions of pure olivine powders and E. coli cells in pure water were also used for reference. Olivine controls reproduce the Mg, Si and Fe release in solutions predicted from rates published in the literature with pH shifts of less than 0.5 unit. After 1 week, under abiotic conditions, weathered surfaces are enriched in Fe and Fe³⁺ relative to the initial composition of the mineral. Bacterial controls (without minerals) show decreasing Eh with increasing cell concentrations (−50 mV with 7 × 10⁷ cells/mL and −160 mV with 8 × 10⁸ cells/mL). Magnesium concentrations in bacterial control solutions are in the μg/L range and can be accounted for by the release of Mg from dead cells. More than 80% of the cells were still alive after 1 week. The solutions obtained in the experiments in which olivine reacts in the presence of cells show Mg and Si concentrations a few tens of percent lower than in the mineral control samples, with a prominent depletion of Fe(III) content of the mineral surfaces. Magnesium mass balance discounts both significant bacterial uptake and inhibition of the Mg dissolution rates as a consequence of changing pH and Eh. Coating by bacterial cell layers is also negligible. E. coli reduces the chemical weathering of olivine. This study infers that the presence of free-living Proteobacteria, a prevalent group of subsurface bacteria, should decrease the amount of riverine Mg released by chemical weathering of mafic rocks.     

Effects of indigenous bacteria on Cr(VI) reduction in Cr-contaminated sediment with industrial wastes

Black, clay-like sediments have been obtained from the area of the pigment manufacturing factories in Dongducheon city, Korea. These sediments were contaminated by heavy metals, especially chromium (700 mg/kg). Indigenous bacteria in the sediments were isolated to investigate their ability to reduce Cr(VI) to Cr(III). The enriched bacterial consortium reduced over 99% of dissolved Cr(VI) in 96 h from the onset of the experiments under anaerobic condition, while there was no change in Cr(VI) concentration until 300 h in abiotic controls. Total amount of dissolved Cr decreased simultaneously when Cr(VI) was reduced, which was likely due to precipitation of Cr(OH)₃ after microbial reduction of Cr(VI) to Cr(III). Under aerobic condition, only 30% of dissolved Cr(VI) was reduced by indigenous bacteria until 900 h. The reduction of Cr(VI) did not accompany bacterial growth since the amount of protein did not show a significant change with time both in the presence and absence of O₂. These indigenous bacteria may play a role in the treatment of Cr(VI)-contaminated sediments.     

Antibiotic resistance and genetic diversity in water-borne Enterobacteriaceae isolates from recreational and drinking water sources

A total of 240 water-borne bacteria including 72 Escherichia coli, 83 Enterobacter, 30 Klebsiella, 36 Salmonella and 19 Shigella spp. isolates from drinking and recreational water sources were assessed for antibiotic resistance and genetic diversity. Escherichia coli (88.89 %) and species of Enterobacter (86.75 %), Klebsiella (83.33 %) and Salmonella (100 %) were resistant to cefadroxil, while >94 % Shigella spp. were resistant to cefaclor and cefuroxime. Ofloxacin was the most effective antibiotic against isolates of all the genera. Multiple antibiotic resistance index identified dug well, pond and piped water supplies as high risk sources of enteric pathogens. Random amplified polymorphic DNA analysis and restriction fragment length polymorphism of amplified 16S rRNA gene were studied for genetic relatedness of Enterobacteriaceae isolates. Primer P1254 identified 10, 16, 4, 4 and 1 distinct random amplified polymorphic DNA group(s) of E. coli, Enterobacter, Klebsiella, Salmonella and Shigella species, respectively. Unlike random amplified polymorphic DNA, restriction fragment length polymorphism using AluI and HaeIII could not segregate isolates in different genetic profiles. 16S rRNA gene of three Enterobacter spp. strains from different sources with similar restriction fragment length polymorphism but different random amplified polymorphic DNA patterns was sequenced, and identified as Enterobacter hormaechei strains skg0061, 0062 and 0063. The sequence information has been submitted to GenBank (HQ322393-95). Biochemically similar but genetically diverse Enterobacteriaceae members from drinking and recreational water sources exhibited varying antibiotic sensitivity. Contamination of water sources with such multiple antibiotic-resistant enteric pathogens poses threat to human health.     

In situ formation of AgNPs on <Emphasis Type="Italic">S. cerevisiae</Emphasis> surface as bionanocomposites for bacteria killing and heavy metal removal

Novel bionanocomposites, S. cerevisiae–AgNPs, were synthesized by in situ formation of AgNPs on S. cerevisiae surface using fulvic acids as reductants under simulated sunlight. S. cerevisiae–AgNPs were characterized using UV–Vis spectroscopy, scanning electron microscope, transmission electron microscope and Fourier transform infrared spectroscopy. These analyses showed that AgNPs were distributed on the surface of S. cerevisiae. The application of S. cerevisiae–AgNPs in bacteria killing and heavy metal removal was studied. S. cerevisiae–AgNPs effectively inhibited the growth of E. coli with increasing concentrations of S. cerevisiae–AgNPs. E. coli was killed completely at high concentration S. cerevisiae–AgNPs (e.g., 100 or 200 µg mL^?1). S. cerevisiae–AgNPs as excellent heavy metal absorbents also have been studied. Using Cd²⁺ as model heavy metal, batch experiments confirmed that the adsorption behavior fitted the Langmuir adsorption isotherms and the Cd²⁺ adsorption capacity of S. cerevisiae–AgNPs was 15.01 mg g^?1. According to adsorption data, the kinetics of Cd²⁺ uptake by S. cerevisiae–AgNPs followed pseudo second-order kinetic model. Moreover, S. cerevisiae–AgNPs possessed ability of different heavy metals’ removal (e.g., Cr⁵⁺, As⁵⁺, Pb²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Hg²⁺, Ni²⁺). The simulated contaminated water containing E. coli, Cd²⁺ and Pb²⁺ was treated using S. cerevisiae–AgNPs. The results indicated that the bionanocomposites can be used to develop antibacterial agents and bioremediation agents for water treatment.     

Isolation and identification of cadmium- and lead-resistant lactic acid bacteria for application as metal removing probiotic

The purpose of the present study was to isolate and identify the metal-resistant lactic acid bacteria from sediments of coastal aquaculture habitats for removal of cadmium and lead from ambience. Collected sediment samples were used to isolate the cadmium- and lead-resistant bacterial colonies by spread plate technique using agar media (De Man, Rogosa and Sharpe) supplemented with cadmium or lead at 50?mg/l. Isolates were identified by bacterial colony polymerase chain reaction and sequencing of 16S ribosomal deoxyribonucleic acid. Metal removing probiotic was determined by characterizing the lactic acid yield in culture media, viability in fish intestine, metal-resistant and metal-removal efficiencies. 16S ribosomal deoxyribonucleic acid sequencing data of five (Cd10, Cd11, Pb9, Pb12 and Pb18) and other all isolates clearly showed 99?% similarities to Enterococcus faecium and Bacillus cereus, respectively. The Pb12 exhibited higher lactic acid yield (180?mmol) than that of the remaining E. faecium strains and excellent viability without pathogenicity; therefore, further study was carried out using Pb12 strain. The selected Pb12 strain showed elevated metal resistant (minimum inhibitory concentrations 120 and 800?mg/l for cadmium and lead, respectively) and removal efficiencies [Cadmium 0.0377?mg/h/g and lead 0.0460?mg/h/g of cells (wet weight)]. From the viability and metal removal points of view, it can be concluded that isolated metal-resistant E. faecium Pb12 strains might be used as potential probiotic strains for removing heavy metals from fish intestinal milieu to control the progressive bioaccumulation of heavy metals in the fish.     

Anoxygenic phototrophic bacterial diversity within wastewater stabilization plant during ‘red water’ phenomenon

The molecular diversity of the purple photosynthetic bacteria was assessed during temporal pigmentation changes in four interconnected wastewater stabilization ponds treating domestic wastewater by denaturant gel gradient electrophoresis method applying pufM gene. Results revealed high phylogenetic diversity of the purple phototrophic anoxygenic bacteria community characterized by the presence of the purple non-sulfur, purple sulfur, and purple aerobic photosynthetic anoxygenic bacteria. This phototrophic bacterial assemblage was dominated by the purple non-sulfur bacteria group (59.3 %) with six different genera followed by the purple sulfur community (27.8 %) with four genera and finally 12.9 % of the pufM gene sequences were assigned throughout the aerobic anoxygenic phototrophic bacterial group. The purple phototrophic bacterial community was characterized by the presence of salt-dependant bacterial species belonging to the genus Marichromatium, Thiorhodococcus, Erythrobacter, and Roseobacter. The wastewater treatment plant performances were unsatisfactory, and the biological and chemical parameters suggested that the purple photosynthetic bloom was correlated with the eutrophic state.     

Soil bacterial strains with heavy metal resistance and high potential in degrading diesel oil and <Emphasis Type="Italic">n</Emphasis>-alkanes

Four bacterial strains, capable of degrading diesel oil, n-alkanes or hexadecane, were isolated from soils contaminated with petroleum oil and identified. Strains of Pseudomonas sp., Pseudomonas putida TPHK-1 and Pseudomonas aeruginosa TPHK-4, were more efficient in degrading high concentrations of the hydrocarbons than the other two strains, Stenotrophomonas maltophilia TPHK-2 and Acenitobacter sp. TPHK-3. P. putida TPHK-1 exhibited tolerance to very high concentrations of heavy metals such as cadmium, lead, zinc and copper. The innate ability of P. putida TPHK-1, as evidenced by the amplified genes alkB1 and alkB2 that encode alkane hydroxylases, and cat12o and cat23o coding for catechol dioxygenase, in degrading diesel oil in the presence of heavy metals is far greater than that of the strains reported in the literature. Heavy metal tolerance coupled with rapid degradation of hydrocarbons, even at high concentrations, suggests that P. putida TPHK-1 has a great potential in remediating soils contaminated with mixtures of hydrocarbons and heavy metals.     

Potency of constructed wetlands for deportation of pathogens index from rural,urban and industrial wastewater

Pathogen removal is essential for wastewater treatment and its potential reuse in agriculture. Three field-scale wastewater treatment systems consisting of free surface flow were operated around 1.5 years receiving water from urban domestic, rural domestic and industrial sources. The study was conducted to evaluate seasonal performance of constructed wetland systems in removing Escherichia coli, Enterococci and total coliforms under continuous hydraulic flow. Results displayed that all three wetlands gain recognition in removing pathogen load with high removal efficacy till water reaches output ports. Removal efficiencies were even higher, 66–93, 78–92 and 80–94% for E. coli, Enterococci and total coliforms, respectively, within constructed wetlands. Remarkably at shorter temporal scales in CW-A, greater homogeneity of pathogen concentrations was assessed at wetland outlet sites. In outlet ports, results displayed a highly effective removal of E. coli concentration 80–90% (June 2015), 86–92% (October 2015) and 79–92% (February 2016), Enterococci 80–94% (June 2015), 83–94% (October 2015) and 80–94% (February 2016) and total coliforms 85–93% (June 2015), 87–95% (October 2015) and 88–96% (February 2016). Positive correlation was observed between bacterial indicators (E. coli–Enterococci, r = 0.038; p < 0.01 and E. coli–total coliforms, r = 0.142; p < 0.01). Removal of bacterial indicators in constructed wetland was also displayed by PCA in which three-component analysis of variance was 98.39% and showed a clear decrease in measured parameter gradients toward samples from outlet ports. Constructed wetlands provide cost-effective treatment systems for reducing the pathogen load in wastewater in variable agro-climatic conditions and thus improve water quality.     

MI琼脂法同时检测环境水样中总大肠菌群和大肠埃希氏菌

总大肠菌群和大肠埃希氏菌是表征环境水样中粪便污染的两个重要指标。对这两个指标的检测,传统的多管发酵法和滤膜法已经无法满足快速、准确的检测要求。而近年来能够快速测定的固定酶底物技术(DST)已经在国内开始运用,但是由于其检测成本较高,影响了其在基层实验室的推广,MI琼脂法的出现较好地弥补了这个缺陷。本文应用MI琼脂法检测环境水样中总大肠菌群和大肠埃希氏菌,对其方法性能进行了测试和研究,选择国家标准方法——远藤琼脂法作为比较对象,通过菌种加标回收试验比较MI琼脂法和远藤琼脂法的灵敏度差异,通过实际水样的检测验证MI琼脂法和国标法是否具有等效性。结果表明:使用MI琼脂法检测总大肠菌群和大肠埃希氏菌加标样品的平均回收率分别为89.2%和88.8%,分别高于远藤琼脂法的81.0%和81.2%,证明MI琼脂法的灵敏度高于远藤琼脂法。MI琼脂法和远藤琼脂法检测总大肠菌群和大肠埃希氏菌的相关系数分别为0.9627和0.9511,都具有显著相关性,且检测结果没有统计学意义上的差异,证明两种方法检测总大肠菌群和大肠埃希氏菌具有等效性。与国家标准方法相比,MI琼脂法具有灵敏度高、操作简便、检测周期短、检测成本低、可同时检测两个指标等优点,可以替代传统的滤膜法作为检测总大肠菌群和大肠埃希氏菌的新方法。