Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.)

In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.     

Influence of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on ammonia-oxidizing bacteria and archaea in rhizosphere and bulk soil

In agricultural plant production nitrification inhibitors like 3,4-dimethylpyrazole phosphate (DMPP) are used to retard the microbial nitrification process of fertilized ammonium to enhance the nitrogen supply for cultivated crops and to reduce nitrogen losses from the production system. Besides the well-known ammonia-oxidizing bacteria (AOB) it is known for a few years that also ammonia-oxidizing archaea (AOA) are able to perform the first step in nitrification, hence being also a target for a nitrification inhibitor. However, so far no information are available concerning the effectiveness of DMPP and its extent towards AOB and AOA, neither in bulk soil nor in the root-rhizosphere complex. We investigated in a field experiment performed according to agricultural practice the effect of DMPP on the abundance of AOB and AOA two, four and eight weeks after fertilization. We observed impaired abundances of AOB but not of AOA in both soil compartments that were still visible eight weeks after application, possibly indicating a reduced effectiveness of the nitrification inhibitor in our study.     

浊度传感器应用于连续监测污水厂进水悬浮颗粒浓度的研究

为使城市污水处理厂更合理高效的运营,可对进水悬浮颗粒浓度的实时监测来实现。通过对进水水样的悬浮颗粒浓度测量值与相应浊度值建立的关系曲线,达到对污水处理厂进水悬浮颗粒浓度（mg／L）的连续实时监测。文中给出了浊度传感器的校准方法,并建立浊度-悬浮颗粒浓度关系曲线,将其应用于某城市污水处理厂,得到单位时间进水悬浮颗粒总量的实时连续图。     

活性污泥总DNA提取方法的比较

从处理某制药废水的MBR反应器中采集活性污泥,评价不同DNA提取方法对其总DNA提取效率的影响。DNA提取分细胞裂解和DNA纯化2步,对细胞裂解比较了珠磨匀浆法、反复冻融法、十二烷基磺酸钠（sodium dodecyl sulfate,SDS）裂解法等7种方法;对DNA纯化比较了酚/氯仿纯化法和胶回收纯化法。结果表明,SDS裂解法、酚氯仿纯化法最优。通过条件优化实验,确定SDS裂解酚/氯仿纯化法在污泥量1.1 g,10 000 r/min离心5 min的操作条件下,获得的DNA产量（10 774 μg/g泥重）和纯度（OD₂₆₀∶OD₂₈₀=1.84）等综合指标最好。     

Characterization of bacterial diversity in an atrazine degrading enrichment culture and degradation of atrazine,cyanuric acid and biuret in industrial wastewater

An enrichment culture was used to study atrazine degradation in mineral salt medium (MSM) (T1), MSM+soil extract (1:1, v/v) (T2) and soil extract (T3). Results suggested that enrichment culture required soil extract to degrade atrazine, as after second sequential transfer only partial atrazine degradation was observed in T1 treatment while atrazine was completely degraded in T2 and T3 treatments even after fourth transfer. Culture independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique confirmed selective enrichment of genus Bacillus along with Pseudomonas and Burkholderia. Degradation of atrazine/metabolites in the industrial wastewater was studied at different initial concentrations of the contaminants [wastewater-water (v/v) ratio: T1, 1:9; T2, 2:8; T3, 3:7; T4, 5:5 and T5, undiluted effluent]. The initial concentrations of atrazine, cyanuric acid and biuret ranged between 5.32 and 53.92 µg mL^?1, 265.6 and 1805.2 µg mL^?1 and 1.85 and 16.12 µg mL^?1, respectively. The enrichment culture was able to completely degrade atrazine, cyanuric acid and biuret up to T4 treatment, while no appreciable degradation of contaminants was observed in the undiluted effluent (T5). Inability of enrichment culture to degrade atrazine/metabolites might be due to high concentrations of cyanuric acid. Therefore, a separate study on cyanuric acid degradation suggested: (i) no appreciable cyanuric acid degradation with accumulation of an unidentified metabolite in the medium where cyanuric acid was supplemented as the sole source of carbon and nitrogen; (ii) partial cyanuric acid degradation with accumulation of unidentified metabolite in the medium containing additional nitrogen source; and (iii) complete cyanuric acid degradation in the medium supplemented with an additional carbon source. This unidentified metabolite observed during cyanuric acid degradation and also detected in the enrichment culture inoculated wastewater samples, however, was degraded up to T4 treatments and was persistent in the T5 treatment. Probably, accumulation of this metabolite inhibited atrazine/cyanuric acid degradation by the enrichment culture in undiluted wastewater.     

Improving Anammox start-up with bamboo charcoal

Three Upflow Anaerobic Sludge Blanket (UASB) reactors were compared for Anammox enrichment using synthetic wastewater with Spherical Plastic (SP) and Bamboo Charcoal (BC) addition, and without carrier (CK). After four months of operation, the Anammox activity occurred in all reactors allowing continuous removal of ammonium and nitrite. Ammonium and nitrite removal efficiencies were all higher than 98% in steady phase with the effluent concentrations below 1 mg L⁻¹. The start-up time could be shortened from 117 to 97 d in CK and SP reactor to 85 d in BC amendment reactor. Quantitative PCR (q-PCR) analyses indicated a significant increase in the number of Anammox bacteria in BC amended reactor as compared with CK and SP during the entire start-up periods. The copy numbers of Anammox of 16S rRNA gene in the reactor with BC amendment could reach up to 6 × 10⁹ copies g⁻¹ Volatile Suspended Solids, around 22.5 times and 12.3 times greater than that in CK and SP reactor, respectively. BC addition could accelerate the start-up of Anammox and significantly increase the Anammox bacteria number.     

水中抑制物对定量PCR与膜过滤法检测E.coli的影响

针对水中病原微生物的污染,选择大肠埃希氏菌E.coli作为病原示踪剂,以E.coli染色体上β-葡萄糖醛酸酶uid A目的基因建立了SYBR Green实时荧光定量PCR的检测方法。该定量PCR方法灵敏度高,检测限可达104CFU/L,线性关系良好,相关系数为R2=0.999。研究表明,定量PCR与菌液浓度呈显著正相关R2=0.935。通过人工投加腐殖酸、COD,研究了水中抑制物对定量PCR和膜过滤MF培养法的影响。结果显示,腐殖酸可使E.coli在培养皿上的菌落变小聚集,且显色不明显,当腐殖酸量为20 mg/L时,浓度为50 CFU/100 m L的E.coli完全受到抑制,没有菌落出现。腐殖酸对PCR扩增的抑制作用明显,当腐殖酸浓度增加到10 mg/L,定量PCR检测结果基因拷贝数减少约1 log,增加到20 mg/L定性PCR检测结果为阴性。     

Detection of Listeria monocytogenes in ready-to-eat food by Step One real-time polymerase chain reaction

The aim of this study was to follow contamination of ready-to-eat food with Listeria monocytogenes by using the Step One real time polymerase chain reaction (PCR). We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Listeria monocytogenes Detection Kit for the real-time PCR performance. In 30 samples of ready-to-eat milk and meat products without incubation we detected strains of Listeria monocytogenes in five samples (swabs). Internal positive control (IPC) was positive in all samples. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in ready-to-eat food without incubation.     

A Comparison of Swine Wastewater Treatment Using Real-Time and Fixed-Time Two-Stage Sequencing Batch Reactors

Abstract

This study compared the swine wastewater treatment of two identical lab-scale two-stage sequencing batch reactors (TSSBR) under similar conditions except that one was operated on a fixed-time mode and the other on a real-time mode. While both TSSBR systems performed very well, the real-time TSSBR performed far better then the fixed-time TSSBR, in every aspect of carbon, nitrogen, and phosphorous removal. The removals of COD, TOC, were at 97% and for BOD₅ even at 99.7%. In terms of NH₄-N and TKN removals, the real-time TSSBR achieved removal of over 98%. For phosphorus removals (Ortho-P and total P) the results from the real-time TSSBR was quite remarkable at 97.7%.     

The Fate of the Recombinant DNA in Corn During Composting

Abstract

In order to make regulations that safeguard food and the environment, an understanding of the fate of transgenes from genetically modified (GM) plants is of crucial importance. A compost experiment including mature transgenic corn plants and seeds of event Bt 176 (Zea mays L.) was conducted to trace the fate of the transgene cryIA(b) during the period of composting. In bin 1, shredded corn plants including seeds were composted above a layer of cow manure and samples from the corn layer were collected at intervals during a 12-month period. The samples were tested for the transgene persistence and microbial counts and also the compost was monitored for temperature. In bin 2, piles of corn seeds, surrounded by sheep manure and straw, were composted for 12 months. A method combining nested polymerase chain reaction (PCR) and southern hybridization was developed for detection of the transgene in compost. The detection sensitivity was 200 copies of the transgene per gram of dry composted corn material. Composting commenced on day 0, and the transgene was detected in specimens from bin 1 on days 0 and 7 but not on day 14 or thereafter. The transgene in corn seeds was not detectable after 12 months of composting in bin 2. Temperatures in both bins rose to about 50°C within 2 weeks and remained above that temperature for about 3 months, even when the ambient temperature dropped below ?20°C. Extracts from compost were inoculated onto culture plates and then were incubated at 23 to 55°C. Within the first 2 weeks of composting in bin 1, the counts of bacteria incubated at 55°C increased from 3.5 to 7.5 log ₁₀, whereas those incubated at 23°C remained at about 7.5 log ₁₀. The counts of fungi incubated at 45°C increased slightly from 2.5 to 3.1 log₁₀, but those incubated at 23°C decreased from 6.3 to 3.0 log ₁₀. The rapid degradation of the transgene during composting of Bt corn plants suggested that the composting process could be used for safe disposal of transgenic plant wastes.