Comparison of metabolic nitramine degradation by denitrifying species

The biodegradation of two popular nitramine energetics were investigated. The HMX (octa‐hydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine) was mineralized by anaerobic mixed denitrifiers in digested sewage sludge culture. An initial HMX concentration of 120 mg/L decreased to a non‐detectable level (> 99% removal efficiency) in 8 days of incubation under strict anaerobic conditions. It was, however, not effectively metabolized by single denitrifying species, P. aeruginosa, B. subtilis in a nitrogen limiting condition under their optimum growth conditions. The other nitramine energetic, ADN (ammonium dinitramide), was mineralized well in the anaerobic mixed culture. The initial ADN concentration of 250 mg/L was reduced to non‐detectable levels (> 99% removal efficiency) in 5 days of incubation under anaerobic conditions. These results show that the anaerobic mixed culture, compared to the pure monoculture, is superior in the degradation of nitramine energetics.     

Degradation of ammonium dinitramide (ADN) in digested sewage sludge under strict anaerobic conditions

The biodegradation of one popular nitramine energetics, ammonium dinitramide (ADN) by mixture of denitrifying bacterial species was investigated. ADN was observed to be effectively mineralized in the anaerobic mixed culture. The initial ADN concentration of 250 mg/L was reduced to non‐detectable levels (> 99% removal efficiency) in 5 days of incubation under anaerobic conditions. Final products generated from anaerobic degradation of nitramine energetics by anaerobic metabolism were NH₄ ⁺, CH₄, and CO₂ that were released to the environment with the denitrifiers’ growth. In addition, it was found that the activity of denitrifiers was inhibited by high concentration of ammonia generated through the degradation reactions of energetic nitrites.     

污泥堆肥利用中AM对稗草生长及Cu、Pb吸收的影响

在滨海盐渍土中分别混加质量分数为0%、20%、40%和60%的污泥堆肥,作为盆栽基质,研究摩西球囊霉（Glomusmosseae）和根内球囊霉（Glomus intraradices）2种AM真菌对稗草（Echinochloa crusgalli）生长及其吸收Cu、Pb的影响,分别以不接种AM真菌的处理为各自的对照。结果显示：添加污泥堆肥处理中稗草接种苗菌根侵染率均显著高于纯盐泽土处理。同时,随着盐渍土中污泥堆肥含量增加稗草生物量上升,其中在含有40%和60%污泥堆肥处理中接种AM真菌稗草的地上及地下部生物量显著高于未接种苗。接种AM真菌显著提高了稗草Cu、Pb富集总量;接种AM真菌显著提高了稗草地下部Cu富集量,却降低了地下部Pb累积量,提高了Pb向地上部的转运,增加了地上部Pb累积量。这些结果表明污泥堆肥中接种AM真菌可以促进稗草的生长和提高对重金属Cu、Pb的富集能力。     

Heavy metals in contemporary Chinese sewage sludges

The heavy metal content in sewage sludges from a big Chinese city was investigated. Concentrations of zinc (Zn), lead (Pb), copper (Cu), chromium (Cr), nickel (Ni), cadmium (Cd) and mercury (Hg) in the sludges were 258–4050 mg kg^‐1, bd: 994 mgkg^‐1, 8.3–566 mg kg^‐1, 26.3–370mgkg^‐1 4.2–113 mg kg^‐1 0.9–6.4 mg kg^‐1 and 1.8–12.4 mg kg^‐1 respectively. The concentrations of Zn and Pb in the sewage sludges from the residential areas were higher than those in the mixed ones (from both residential and industrial areas). The concentrations of heavy metals in the flocculently dewatered sewage sludges were higher than those in the sediment of the centrifuged undewatered sewage sludges. After centrifuging, more than 60% of heavy metals remained in the sludge sediment with an exception of Cd. The content of organic matter, total phosphorus (T‐P) and total potassium (T‐K) in these sewage sludges was also measured.     

Effect of poultry,cattle, sheep manures and sewage sludge on N mineralisation

The effects of various organic wastes on nitrogen mineralisation in soil were investigated. For this purpose, poultry (pl), cattle (ct), sheep (sh) manures and sewage sludge were used as organic wastes. This study was conducted as a laboratory incubation experiment with a calcareous soil. Organic wastes (3%) were added to pots of soil and incubated at 28°C for 16 weeks under non-leaching conditions. The cumulative mineralised N was then fitted to a single exponential model. Maximum nitrogen mineralisation was determined in the second week of incubation in soil treated with poultry manure. Overall, the results showed that the amount of mineral nitrogen in soil treated with different organic wastes was controlled by the type of manure. The results indicated that among the organic wastes, sewage sludge induced the highest quantities of net N mineralisation. Generally, organic wastes increased the amount of N mineralisation in the studied soil and the values of N₀ and k in treated soil varied depending on the type of organic waste. The highest N₀ and k values were found in poultry-treated soil.     

Robin’s egg blue: does egg color influence male parental care?

A recent hypothesis suggests that birds’ blue-green egg colors may be a sexually selected signal of female (and potentially nestling) quality that males use to make parental investment decisions. While there is some empirical support for this idea, both theory and observations question its validity. To test this hypothesis experimentally, we examined the influence of egg color on male American robin Turdus migratorius behavior by replacing natural clutches with four artificial eggs that were all either pale or vividly colored, close to the extremes in natural egg coloration. At the end of the incubation period, three unrelated nestlings were fostered into each experimental nest, and parental provisioning behavior was monitored when nestlings were 3, 6, and 9 days old. Male provisioning rate for 3-day-old nestlings was significantly higher in the vivid egg treatment compared to both the pale egg treatment and untreated controls, but there was no effect of egg color on paternal behavior at the older nestling stages. Male feeding rate at unmanipulated nests was only weakly positively related to natural egg color (chroma) when nestlings were 3 days old. These results suggest that blue-green egg color may act as a post-mating signal of female quality or investment in this species, but our findings do not exclude the possibility that egg color pigmentation also serves other adaptive functions.     

Survival of faecal coliforms and hygiene risks in soils treated with municipal sewage sludges

An incubation experiment was conducted to monitor effect of sewage sludge application on changes in numbers of faecal coliforms in soils over time after sludge application and evaluate the hygiene risks. Soil faecal coliform counts were made after 1, 7, 14, 28, 56 and 84 days of incubation. The faecal coliform counts in the sludge-treated soils decreased substantially with time and were similar to those in the untreated controls after incubation for 56 days. Land application of air-dried sludges increased the hygiene risks due to the re-growth of faecal coliforms, and the counts of faecal coliforms in soil treated with air dried sludge from Suzhou (91% DM) were 50 times higher than in soils with fresh dewatered sludge from Suzhou (15% DM) after 7 days of incubation. The main factors affecting the changes in faecal coliform counts were sludge type and incubation time. Sludge type determined the faecal coliform counts and the ability of the faecal coliforms to re-establish, and indigenous microorganisms competed with the faecal coliforms for nutrients during the incubation process.     

A modified method for determination of dehydrogenases activity of activated sludge

Examination of the different methods employed to detect dehydrogenases activity of activated sludge by reduction of triphenyl tetrazolium chloride (TTC) indicated the non‐uniformity of the procedures. A modified method is, therefore, developed which consists of incubation of a dilute sonicated sample of mixed liquor suspended solids (MLSS) of activated sludge with TTC in the presence of sodium sulfite, phosphate buffer and cyanide. Triphenyl formazan (TF) formed is first dissolved in acetic acid and later extracted by toluene. Optical density (OD) of the color is measured at 485 nm. Dehydrogenases activity is calculated from the OD and expressed as specific activity per unit weight of protein. Modified method gave less relative dispersion compared to the values obtained on the same but unsonicated samples of MLSS.     

Influence of phytoplankton on complexing capacity of seawater†

Differential Pulse Anodic Stripping Voltammetry (DPASV) was employed to measure Apparent Copper Complexing Capacity (L_t) and Stability Constants (K_s) of related cupric complexes of cell exudates of the phytoplanktonic alga Dunaliella salina. Three natural seawater samples collected 0.1, 2, 5 miles off the Tyrrhenian coast were used as culture medium. The measurements were carried out 1h and 7 days after the inoculum. Furthermore, on one culture the medium was passed through C₁₈‐SEP‐PAK cartridges in order to separate the cell exudates. The results show that Dunaliella salina influences the value of L_t but does not affect the value of K_s . The C₁₈SEP‐PAK retain only 50% of the ligands produced by the cells and capable of complexing copper.     

Catalase activity of activated sludge

Catalase activity of mixed liquor suspended solids of aeration tank of activated sludge plants in Milwaukee, WI, was measured in sonicated samples. Catalase activity varied in the samples of mixed liquor suspended solids collected from the head and end of the aeration tanks; in return activated sludge; and in waste activated sludge. Catalase activity decreased in mixed liquor suspended solids, on prolonged aeration (8 days), in the absence of added sewage. Volatile suspended solids and catalase activity seemed to bear a relationship.     

Anaerobic degradation kinetics of reactive dye with different carbon sources

This study aims to investigate the anaerobic degradation kinetics of reactive dye, C.I. Reactive Red 141 (Evercion Red H-E7B) by partially granulated anaerobic mixed culture using three carbon sources, namely modified starch (MS), polyvinyl alcohol (PVA) and acrylic size (AS) during batch incubation. There is a first-order kinetics reaction in the decolorization processes using MS and PVA as carbon sources, while a zero-order kinetics relationship describes the decolorization process for the AS carbon source. The k values and color removal rate of decolorization with MS carbon source was higher than those of PVA and AS carbon sources. This is because the MS carbon source was well degraded in comparison to AS and PVA, respectively This study also found dye reduction could be enhanced through the addition of MS as a carbon source. The decolorization rates increased with decrease in dye concentrations of RR 141. In contrast, the decolorization rates increased with increase in COD concentration.     

Uptake and genotoxicity of micromolar concentrations of cobalt chloride in mammalian cells

Literature data concerning the genotoxicity of cobalt salts have been conflicting. To establish appropriate incubation conditions, we conducted a series of uptake studies, before genotoxicity was determined by DNA strand break induction in HeLa cells and mutagenicity in V79 Chinese hamster cells. Co(II) is taken up by HeLa cells in a concentration‐dependent manner and is accumulated inside the cell. The uptake is preceded by a fast association step to the outer membrane, with no saturation up to 24 h. DNA strand breaks as determined by nucleoid sedimentation are induced at concentrations as low as 50μMCoCl₂. The induction is time‐dependent, showing the highest number of breaks after 4h incubation with no further increase up to 24h. CoCl₂ is mutagenic at the HPRT‐locus, enhancing the spontaneous mutation frequency 4.2‐fold at 100μ?. Besides direct interactions with DNA, the mutagenicity of CoCl₂ could also be due to a decrease in the Fidelity of DNA polymerisation.     

Potentiometric studies of mixed complexes of cobalt(II) and copper(II) with l‐Asparagine and adenine

A potentiometric titration technique has been used to determine the stability constants for the various complexes of Co(II) and Cu(II) with L‐asparagine and from DNA base, e.g. adenine. Stability constants of ternary systems have been evaluated by the method suggested by Irwing‐Rossotti. In addition, the conditional constants were calculated as a function of pH. The maximum values of the conditional formation constants were found to be in accordance with the mixed‐ligand complex formation constants in a determined pH region. Furthermore, the molar fractions of different species from mixed complexes were calculated by means of formation constants. The values of stability constants of mixed‐ligand complexes at 25°C are as follows: log K= 5.25 for Co(II)‐L‐asparagine‐adenine; log K= 9.30 for Cu(II)‐L‐asparagine‐adenine. The ionic strength was kept constant at I = 0.20 with NaClO₄.     

Life history of a free-living marine nematode Daptonema normandicum reared in laboratory

Life history of a free-living meiobenthic nematode Daptonema normandicum (DeMan, 1890) was studied in the laboratory. Live specimens were primarily collected from the sewage outlet site near the mouth of the Mandovi estuary, Goa This species was the most dominant (> 67%) among the meiobenthic nematodes. Vertically, nematode abundance was highest at the surface sediment and correlated with the organic carbon and sediment chlorophyll-a. Considering their dominance in the meiofauna, attempts were made to rear D. normandicum in laboratory. Salinity of the culture medium was maintained at 14 to 17 PSU (same as the collection site). All the culture experiments were conducted in semisolid nutrient agar media at 27 +/- 2 degrees C temperature for 12 hr dark: 12 hr light conditions. The food consists primarily of an unidentified bacterium and mixed algae, but diatom and ciliates were also observed in culture. Females produced first batch of eggs at the age of 23 days. Gravid female normally carry 8-10 eggs. Embryonic development is completed in -72 hr and entire life cycle (egg to adult) was completed in 22-24 days. Average size of juveniles at the hatching was 0.189 mm. Young individuals attains a maximum size of 1.23 mm (male) and 1.04 mm (female) in -21-23 days. Growth, in terms of length was augmented upto 23rd day and ceased thereafter. The daily growth increment for the first 5 days was 0.01-0.04 mm which increased upto 0.05-0.08 mm d(-1) during the maturation (10-18 days). Male : female ratio was 1:2. In this laboratory study, we provided information on the embryonic development, the life cycle and ecology Our results demonstrated that D. normandicum can be reared successfully under the controlled conditions, suggesting possible use of this species in toxicological and aquaculture studies. The culture method described is very handy and can be applicable for rearing other meiobenthic species particularly the nematodes with comparable feeding habits.     

Binding of trinitrotoluene (TNT) to water extractable humus

Solid-state ¹⁵N NMR was applied to the aqueous extracts of a ¹³C-enriched plant slurry (Lolium perenne), anaerobically incubated with ¹⁵N₃-trinitrotoluene (TNT). Almost all ¹⁵N₃-TNT transformation products became covalently bound to the plant-derived organic material extractable with water. DCPMAS ¹⁵N ¹³C NMR revealed a three-step reaction scheme. After reduction of TNT, the aryl amines are acetylated. Subsequent alkylation of the resulting amides strengthens the incorporation of TNT-transformation products into humic material. Comparable results have been recently obtained under aerobic conditions, which indicates that this pathway is a common process during biological TNT transformation.     

Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M₁ (AFM₁) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM₁ sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM₁ in milk. However, if there was no color change, presence of AFM₁ at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM₁ detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM₁ in milk.     

Studies on the causes of mortality of the estuarine bivalve Macoma balthica under conditions of (near) anoxia

Survival of the bivalve Macoma balthica in (near) anoxic seawater was studied in a static system and a flow-through system and compared with emersed exposure to air and N₂. In the static system, a decrease in pH and exponential accumulation of sulphide in the incubation medium were observed, indicating excessive growth of (sulphate-reducing) bacteria. These changes in the chemical environment were prevented by the use of a flow-through system. However, this treatment hardly affected survival time. Median mortality times were 8.3 and 9.0 days for the static and flow-through incubation, respectively. Addition of the antibiotic chloramphenicol strongly increased survival time in both systems with corresponding values of 17.9 and 23.0 days. A similar value was obtained for survival in air (LT₅₀= 21.7 days). In a second experiment (1 year later), we obtained much lower values for anoxic survival in a static system, although laboratory conditions, season and temperature were similar. The pH values were adjusted to 6.5, 7.2 and 8.2 by buffering the media (25 mM Tris-HCl), and the corresponding LT₅₀ values were 5.5, 5.7 and 4.7 days, respectively. In the presence of chloramphenicol the values were 10.8, 10.9 and 9.5 days, respectively. These values show that a slightly acidic medium increased survival time. Exposure to an atmosphere of N₂ resulted in a survival time close to that in anoxic seawater without chloramphenicol (LT₅₀= 6.4 days). Overall the results indicate that proliferation of anaerobic bacteria associated with the bivalves was the main cause of death. Since chloramphenicol also displayed a strong positive effect in the flow-through system, which prevented the accumulation of released waste products and a decrease of pH, bacterial damage must have been by injury of the tissues of the clams and not by the release of noxious compounds to the medium. Bacterial outbreaks are a part of every anoxic event (eutrophication), and therefore, in their habitats, direct bacterial infection may also be the cause of clam mortality. It is concluded that laboratory studies on anoxic tolerance, or impact of sulphide, may produce artefacts when no precautions are taken to suppress bacterial proliferation. Received: 4 July 2000 / Accepted: 6 December 2000     

Degradation of crude oil by a mixed population of bacteria isolated from sea-surface foams

A mixed bacteria population (EM₄) was isolated from foams formed on the surface of a zone chromically polluted by hydrocarbons (Gulf of Fos, French Mediterranean coast, October 1981). The population was able to degrade crude oil very effectively in the presence of sea water supplemented with nitrogen, phosphorus and iron. The percentage of hydrocarbon degradation was 81% at 30°C, pH 8, and partial oxygen pressure of 100%. After 12 d incubation, 92 and 83% of satured and aromatic compounds (mono-, di- and triaromatics) were degraded, respectively, as well as 63% of polar products and 48.5% of asphaltenes. Maximum degradation was attained at a sodium chloride concentration of between 400 and 800 mM with Population EM₄, which is constituted of 8 strains, four of which are weak halophiles. Bacterial growth on hydrocarbons induces the production in the culture medium of surface-active agents which are able to emulsify the substrate. There is high specificity between the nature of the growth substrate and such emulsifying activity, particularly as far as petroleum is concerned: only the culture medium from Population EM₄ is able to emulsify petroleum. These surface-active agents contain sugars and lipids (fatty acids, mono- and diglycerides). The foams, which always contain a high concentration of both hydrocarbons (100 to 180 mgl^-1) and bacteria that are able to grow on these types of substrates, have a strong emulsifying activity. Our results would seem to demonstrate the importance of biosurfactants in the elimination of hydrocarbons from polluted biotopes.     

Phytotoxicity to and uptake of TNT by rice

The contamination of the environment by explosives is a worldwide problem resulting in part from 2,4,6-trinitrotoluene (TNT) production. In situ phytoremediation is an appropriate, alternative, cost-effective technology to detoxify extended contamination of surface soil. The ability of rice (Oriza sativa) to both tolerate and assimilate ¹⁴C-labeled TNT was investigated over a 40-day exposure period. The germination rate decreased at 500 mg/kg TNT whereas root and shoot length increased significantly at high TNT concentrations, from 150 to 500 mg/kg. Rice took up TNT residues from soil and accumulated most in roots. Less than 25% of radioactivity taken up was translocated to aerial parts. Above 200 mg/kg TNT, the concentration of TNT residues in roots reached a maximum of approximately 0.7 mg/g. No TNT was found in plant extracts, good evidence for rapid metabolism of TNT. More than 60% of ¹⁴C activity was found as unextractable residues in roots. It was concluded that TNT metabolized and subsequently sequestered by roots could not be translocated to aerial parts.     

农用污泥中铜的生态安全阈值研究

本论文采用污泥和土壤外源铜的活性差异系数法研究了农用污泥中铜的生态安全阈值。首先通过比较确定不同土壤条件下污泥中铜和水溶性铜的活性差异的系数,然后利用系数法得到相应土壤条件下污泥铜HC5值(即能够保护95%物种的浓度),并建立了土壤理化性质参数与污泥铜HC5值的量化关系和预测模型。最后与我国现行污泥农用标准值进行了比较,提出了修改建议。结果表明,土壤中来源污泥的铜活性均值约为来源水溶性盐的40%左右。土壤阳离子交换量(CEC)是影响土壤中污泥源铜的毒性的主控因子,可影响污泥铜HC5值变异的84.6%,而土壤pH值和有机碳含量(OC)分别可影响污泥铜HC5值变异的8.4%和1.8%。基于土壤pH、OC和CEC的三因子模型进行预测优于基于土壤pH和CEC的两因子模型,其相关性达到94.8%。与模型预测值相比,我国现行污泥农用标准GB4284-84与CJ/T309-2009中对铜限值的规定均存在着不同程度的保护不足或保护过度问题。