A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors

Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus, the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte species to overcome major limitations for use. Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes, and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical. Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69 mg/L for paraquatdichloride, alizarine and triclosan, respectively. Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time (0.026 mg/L) as well as with literature findings. Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however, the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might be due to physicochemical substance properties such as lipophilicity. Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation, or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary. ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com)     

DO和pH值在短程硝化中的作用

在SBR反应器中对DO和pH值在短程硝化和半亚硝化过程中的作用进行试验研究,结果表明,控制低DO和适宜的pH值在短程硝化过程中起着重要的作用.本试验条件下,当DO为0.5～1.0 mg/L、pH值为7.5～8.0时,在SBR反应器中很容易实现短程硝化;当DO＞0.3 mg/L时,DO越低,出水NO2--N积累率越高;当pH值＞6.8时,不会影响系统NO2--N积累的稳定性.另外,研究结果还表明,通过控制DO和pH值可以实现半亚硝化.本试验条件下,当进水氨氮浓度为120 mg/L时,控制DO为0.3～0.4 mg/L可实现出水半亚硝化;当进水氨氮浓度为200 mg/L时,控制DO为0.5～0.6 mg/L或pH值为6.8也可以实现出水半亚硝化.     

反硝化除磷的影响特性试验

采用SBR反应器（厌氧/缺氧/好氧工艺）,分别研究了乙酸盐及硝酸盐浓度变化对反硝化除磷的影响特性.试验结果表明,当进水COD浓度＞230 mg/L时,乙酸盐浓度的变化对释磷、除磷速率等影响并不显著.在硝酸盐浓度＜30 mg/L时,硝酸盐浓度越高,缺氧段除磷速率也就越高.在C/P＞23,C/N＞5条件下,SBR系统对磷、氮去除率在90%以上.     

ASBR中强化活性污泥与普通活性污泥降解啤酒废水的对照研究

在厌氧序批式反应器(ASBR)中采用优势菌群对活性污泥进行强化，驯化出强化活性污泥。与普通活性污泥处理啤酒废水对照显示，强化活性污泥较普通厌氧污泥驯化成熟时间短，处理效果更稳定、更有效，CODcr去除率最高达95％。培育出的强化活性污泥颗粒粒径为1．5～2．5cm，较普通活性污泥颗粒粒径更大，更均匀。     

碳氮磷比例失调城市污水的同步脱氮除磷

为解决现行同步脱氮除磷工艺处理南方地区碳、氮、磷比例失调城市污水中,因C/N、C/P偏低,碳源不足而降低脱氮除磷效率的难题,试验以碳源偏低的广州市城市污水为研究对象,采用厌氧/好氧交替运行的SBR系统,通过对厌氧、好氧时段的合理调控,在无需额外添加碳源的条件下,有机物、氨氮、总氮和总磷的平均去除率分别可达90%、72%、41%和99%,不仅能使有机物和氮的出水指标达到国家排放标准,而且总磷出水浓度能达0.5 mg/L以下。通过进一步分析同步高效脱氮除磷的影响因素和控制条件,得出合理污泥龄的控制是实现同步脱氮除磷的关键,厌氧/好氧交替运行的方式不仅强化了磷的释放和吸收,而且降低了碳源偏低和硝酸盐对同步脱氮除磷影响的结论。     

有机负荷和溶解氧的变化对SBR污泥膨胀的影响及控制方法

本文研究SBR工艺处理汽车电泳废水在不同有机负荷、溶解氧条件下污泥膨胀的现象 ,分析膨胀发生机理 ,分别提出控制方法。实验结果表明 ,在低、中、高负荷时 ,只要溶解氧发生变化 ,均有可能发生膨胀。低负荷、正常溶解氧时发生的膨胀可以通过前联好氧生物选择器加以控制 ;中负荷、低溶解氧及高负荷、低溶解氧活性污泥发生膨胀时可以通过强化曝气得到控制     

马氏甲烷八叠球菌S－6的一个染色体区段的表达、序列分析和结构分析

用一组多克隆抗体对马氏甲烷八叠球菌（Ｍｅｔｈａｎｏｓａｒｃｉｎａｍａｚｅｉ）Ｓ－６菌株的基因组ＤＮＡ文库进行了筛选．仅选出ｐ６０Ａ克隆能对马氏甲烷八叠球菌中可发生细胞形态学变化菌株的抗血清发生阳性反应．对ｐ６０Ａ克隆的双链ＤＮＡ进行了序列分析．识别出一开式阅读框架（ｏｐｅｎｒｅａｄｉｎｇｆｒａｍｅＰ，ＯＲＦＰ）．表达的蛋白是ＯＲＦＰ编码的蛋白的３倍，并得到ＯＲＦＰ蛋白的三聚体，在ＳＤＳ－ＰＡＧＥ中表现出整体蛋白的迁移行为．同时，此表达蛋白抗１０％ＳＤＳ，６ｍｏｌ／Ｌ尿素及热处理．基因结构分析表明，ＯＲＦＰ具有与Ｍ．ｂａｒｋｒｉｍｃｒＡ有同源性的核糖体结合位点和一个与甲烷细菌启动子共有序列相似度达７７％的启动子序列．使用参照序列分析表明，由ＯＲＦＰ演绎的氨基酸序列，具有高密度的带电荷的氨基酸和占优势的β－层迭构型．对此表达蛋白作了Ｎｅｕｒｏｓｒｏｒａｃｒａｓｓａ的ｐｏｒｉｎ蛋白抗血清试验，以研究其可能功能．检验了Ｍ．ｍａｚｅｉＳ－６细胞的蔗糖梯度制备物，对此原细胞中的ＯＲＦＰ蛋白作了定位．结果表明，ＯＲＦＰ蛋白可能是一种古细菌Ｐｏｒｉｎ，其在Ｍ．ｍａｚｅｉＳ－６中的表达，可能象大肠杆菌那样与渗透压调节有关．     

厌氧氨氧化菌富集培养过程微生物群落结构及多样性

为深入理解厌氧氨氧化菌富集培养过程微生物群落变化特征,采用ASBR反应器进行厌氧氨氧化菌富集培养,考察了不同培养时间微生物群落组成、多样性及物种网络关系.结果表明,通过逐步提高基质浓度,实现了厌氧氨氧化菌富集,NH₄⁺-N和NO₂^--N去除率分别为97.6%和95.4%,总氮去除率为84.9%.高通量测序发现,整个培养过程优势菌门（相对丰度>5%）为变形菌门（Proteobacteria）、拟杆菌门（Bacteroidetes）、绿弯菌门（Chloroflexi）、浮霉菌门（Planctomycetes）、装甲菌门（Armatimonadetes）和放线菌门（Actinobacteria）;富集培养获得的主要厌氧氨氧化菌为Candidatus Brocadia,相对丰度从1.42%增长到24.66%;培养过程,微生物群落优势菌群组成未发生变化,但相对丰度呈现显著差异（P<0.05）.富集培养过程不同时间,微生物群落α多样性呈现先升高后降低的趋势,且存在显著差异（P<0.05）;微生物群落β多样性在富集培养过程发生明显空间分异特征,且存在显著差异（R=0.5672,P<0.01）.培养过程不同时间,物种网络密度分别为0.188、0.068、0.059、0.18和0.0735;虽然富集培养过程导致微生物间的关联作用变弱,但浮霉菌门相关类群的物种成为网络中的主要节点.     

进水模式对SBBR性能及氮形态转化的影响

通过对4种不同进水模式下序批式生物膜反应器(SBBR)的性能、微生物群落结构以及氮形态转化的差异分析, 比较不同进水模式对SBBR性能和氮形态转化的影响及其产生的机制. 结果表明, 分散式进水模式表现出比一次性进水更好的脱氮效率和更高的抗冲击负荷能力, 在达到相同的处理效率的前提下, 分散式进水模式M₄的COD和氨氮负荷最高可达2 540和540 mg·(L·d)^-1, 而一次性进水模式M₁仅能分别达到2 000和420 mg·(L·d)^-1;分散进水模式能降低一次性进水所带来的冲击性负荷, 将负荷均化分散到周期内的各个时段, 同时也减少了进水对微生物的稀释作用, 使得单位体积内有效微生物的数量相对充足, 从而提高反应器的负荷能力. 在分散进水模式下, 从M₄与M₂、M₃的对比来看, 分散模式的进水规律越接近运行模式的循环规律, 反应器的氮素转化效率就越高, 残留的氮素总量也就越低.     

基于SBR反应器的ANAMMOX工艺的启动运行

采用SBR反应器,接种好氧硝化污泥,在142 d内于较高负荷下成功启动了厌氧氨氧化反应器.反应器总氮容积负荷(以N计)为0.43 kg/m3·d,总氮去除率最高达到93.3%,平均为80.5%;氨氮和亚硝酸盐氮的去除率最高达到93.9%和99.8%,平均去除率为81.2%和85.7%.在稳定运行阶段,氨氮去除量、亚硝酸盐氮去除量、硝酸盐氮生成量三者之间的比值为1:1.38:0.18.反应器启动过程中,出水、进水pH差值的变化趋势由负到正,然后稳定在一定范围内;且污泥性状有较大变化,污泥中微生物所占比率有所提高,整个反应器中适应厌氧氨氧化运行方式的菌种增殖较快.