Stoichiometric and molecular evidence for the enrichment of anaerobic ammonium oxidizing bacteria from wastewater treatment plant sludge samples

Anammox enrichments were readily developed from seven municipal wastewater treatment plants (WWTPs) sludge, but not with methanogenic granular sludge from two agro-industrial WWTPs. Only 50 d was required for the first evidence of anammox activity from a return activated sludge obtained from a WWTP operated for nutrient removal. The molar ratios of nitrite and ammonium consumption of approximately 1.32 as well as nitrate and dinitrogen gas product ratios of approximately 0.095 provided evidence of the anammox reaction. The presence of anammox was confirmed by polymerase chain reaction (PCR) using primer sets (PLA46F and AMX820R) specific for anammox bacteria. The 16S rRNA gene fragment of anammox bacteria was detected in seven enrichment cultures (ECs) with demonstrated anammox activity but not in the original inocula from which the ECs were derived and also not in the two methanogenic sludge samples, which indicates the PCR predicted the anammox activity. Two genera, Brocadia and Kuenenia, were successfully identified as the Planctomycetes occurring in the clone libraries of successful anammox enrichments. Brocadia dominated in cultures that were respiked extensively; whereas Kuenenia predominated in cultures that were less aggressively respiked. These findings indicate that respiking management may play an important role on selecting the genus of anammox bacteria. The batch enrichment results clearly illustrate that anammox can be readily enriched from municipal sludge from a wide variety of process operations at WWTPs.     

Evaluation of the toxicity of substances to be assessed for biodegradability

The ability to distinguish between true non-biodegradability, and inhibition of biodegradability due to the toxic effects of the compound tested present problems. It results in possible false identification of substances as non-biodegradable.Six chemicals were evaluated in a variety of tests to assess their toxicity to aerobic sludge organisms. The tests employed were: BOD₅ and Closed Bottle inhibition tests, inhibition of respiration of activated sludge, growth inhibition of activated sludge, light emission from (MICROTOX test) and the repetitive die-away test (RDA). Results from these tests were compared with results obtained from a number of ready biodegradability tests using the compounds listed above at anticipated non-toxic and toxic concentrations.No test method evaluated consistently forecast toxicity due to the chemicals tested. The MICROTOX and nitrification inhibition tests were too sensitive. A combination of sludge respiration rate and/or growth tests seem most appropriate. To avoid toxicity to the sludge, biodegradability testing should be made at 10% of the EC₅₀ value. Compounds with an EC₅₀ value greater than 300 mg/l are unlikely to be toxic in ready biodegradability tests. Compounds with an EC₅₀ value of less than 20 mg/l may pose problems necessitating the use of the stringent closed bottle test, or the use of C¹⁴ labelled test materials. Compounds with an intermediate EC₅₀ that is between 20–300 mg/l, need to be evaluated at a range of concentrations in biodegradability tests, or may need to be evaluated carefully to define the precise no-effect level. Procedure recommendations to distinguish between the inhibition of biodegradability and inertness of the test substance are made.     

Evaluation of a serum bottle technique for assessing the anaerobic biodegradability of organic chemicals under methanogenic conditions

The serum bottle method of Shelton and Tiedje (1984), for assessing the potential of an organic chemical to undergo anaerobic biodegradation, was evaluated using U.K. primary digesting sludges. The method was modified by using the sludge at a known solids content and employing sterile (autoclaved) controls to correct for abiotic gas production. Gas production was measured with a compact hand-held pressure meter, which represented an improvement over previously described techniques. Results obtained confirmed the reliability and precision of this type of simple, screening test for assessing anaerobic biodegradability. However, the test is only applicable to low-sulphate, methanogenic environments and evidence is presented of interference from sulphate-reducing bacteria (SRB) when testing sulphate-containing chemicals.     

The anaerobic biodegradability of municipal sludge and fat, oil, and grease at mesophilic conditions.

The anaerobic biodegradability of municipal primary and secondary sludge with increasing levels of partially dewatered fat, oil, and grease (FOG) was assessed using a mixed methanogenic culture at 35 "C. Under batch conditions with an acclimated and enriched microbial population, the sludge loading was 3 kg volatile solids/m3 and the highest FOG loading tested was 1.5 kg volatile solids/m3, resulting in a methane yield of 245 mL methane/g sludge volatile solids added at 35 degrees C and 1010 mL methane/g FOG volatile solids added at 35 degrees C. Under semicontinuous feeding conditions, the sludge and sludge plus FOG loading tested were 3 and 3.75 kg volatile solids/m3-d, respectively. Within 23 days of operation, the volatile fatty acid concentrations were reduced below 200 mg chemical oxygen demand/L (187 mg/L as acetic acid). Enhancement of sludge digestion was observed in those reactors where codigestion of sludge and FOG took place, which was attributed to a higher level of microbial activity maintained in these reactors as a result of FOG degradation. The results of this study demonstrate that beneficial use of FOG through codigestion with municipal sludge is feasible.     

Anaerobic inhibition and biodegradation of antibiotics in ISO test schemes

Municipal sewage is the main exposure route for antibiotics that are used in human medical care. Antibiotics that adsorb to the primary sludge and/or sur-plus activated sludge will enter the anaerobic digesters of municipal sewage treatment plants. Here anaerobic biodegradation or inhibition of anaerobic bacteria resulting in a disturbance of the process might occur. ISO standards 13641 (2003) and 11734 (1999) were used for assessing the anaerobic inhibition of 16 and the anaerobic biodegradability of 9 antibiotics respectively. Digestion sludge from a municipal sewage treatment plant (1g/l d.s.) was used as inoculum in both tests. In ISO 13641 (2003) most antibiotics showed only moderate inhibition effects after a 7 day incubation period, with EC50 values between 24 mg/l and more than 1000 mg/l (equal to mg/g d.s.). In contrast, metronidazol was decisively toxic to anaerobic bacteria with an EC50 of 0.7 mg/l. In the anaerobic degradation tests according to ISO standard 11734 (1995), only benzylpenicillin showed certain ultimate biodegradation after 60 days and most antibiotics inhibited the digesting sludge in the respective parallel tested inhibition controls. Thus the inhibition of anaerobic bacteria by antibiotics observed in the degradation tests was higher than expected from the results of the inhibition tests. The possible explanations are that distinct substrates are used (yeast extract versus sodium benzoate), that the digestion sludge loses activity during the washing steps performed for the degradation tests and that the exposure time in the degradation tests was 8 times longer than in the inhibition test.     

ECOSUNIDE工艺活性污泥中自养菌浓度的测定与分析

设计并利用在差压仪中密闭投加基质的实验方法,根据ASM模型中自养菌和硝化过程耗氧速率的关系,对比研究了ECOSUNIDE工艺和其他4个污水处理厂曝气池混合液中自养菌的浓度。研究表明,在不考虑自养菌增长和衰减情况下,ECOSUNIDE工艺混合液自养菌生物浓度为48.15 mg COD/L,该结果显著高于其他受试污水处理厂的混合液自养菌生物浓度(17.2～37.5 mg COD/L),表明ECOSUNIDE工艺在提高活性污泥中自养菌生物浓度上有较强的优势。设计的实验方法可以简便、有效地测定用于ASM模型中的自养菌浓度。     

城市垃圾焚烧渗沥液中Ca2＋对厌氧EGSB处理效果的影响

研究了城市生活垃圾焚烧厂渗沥液中Ca2＋对厌氧颗粒污泥膨胀床反应器（EGSB）处理效果的影响，并采用静态实验方法考察了Ca2＋对厌氧颗粒污泥产甲烷活性的影响。实验结果表明，进水COD为17000mg／L的条件下，当Ca2＋浓度低于6000mg／L时，EGSB对COD去除率达93％以上；当Ca2＋浓度高于6000mg／L时，COD去除率随运行时间明显下降，并在污泥中形成大量沉淀。静态实验结果表明，废水中低浓度Ca2＋促进了厌氧颗粒污泥的产甲烷活性，但高浓度Ca2＋明显抑制了其产甲烷活性，这是导致高Ca2＋浓度条件下EGSB对COD去除率降低的主要原因。研究表明，颗粒污泥产甲烷活性恢复程度随Ca2＋浓度增加而减弱。     

Effect of the alkyl chain length on the anaerobic biodegradability and toxicity of quaternary ammonium based surfactants

The anaerobic biodegradability and toxicity to methanogenic gas production of different alkyl chain length homologs of quaternary ammonium based surfactants were examinated. Two series of these cationic surfactants were selected: alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium compounds. A simple anaerobic gas production test containing municipal digester solids as a source of anaerobic bacteria was used. Under the applied methanogenic conditions, the cationic surfactants tested showed a very poor primary biodegradation and no evidence of any extent of ultimate biodegradation was observed. The toxicity of quaternary ammonium based surfactants to methanogenic gas production decreased with increasing the alkyl chain length.     

生物降解实验中的接种物标准化研究

针对降解实验中接种物采集困难、活性差异大、实验可重复性及结果可比性差的问题展开接种物标准化研究。对多来源的接种物在实验室可控条件下进行驯养,驯养后生长状态良好的活性污泥分别固定于灭菌麸皮和藻酸钙凝胶等2种微生物载体上,形成2种固定化接种物(分别简称为Bran及Ball)。对上述固定化接种物进行微生物活性、适应性、降解能力以及稳定性研究:通过2种固定化接种物与商品化接种物Polyseed对葡萄糖-谷氨酸(GGA)五日生化需氧量(BOD5)的结果与活菌细胞数的关系,确认3种接种物在降解实验中的用量可控制在相同数量级;比较不同批次及保存时间的2种固定化接种物、相同批次不同保存时间的Polyseed以及污水处理厂曝气池污泥为接种物对苯胺及二甘醇的快速生物降解实验(OECD301F)结果,发现Ball及Bran较Polyseed及新鲜采集活性污泥的微生物适应性及降解能力更佳。此外,在较长保存期内2种固定化接种物活性相对稳定。2种固定化接种物初具成为标准化接种物的必要条件。     

城市垃圾焚烧渗沥液中Ca2+对厌氧EGSB处理效果的影响简

研究了城市生活垃圾焚烧厂渗沥液中Ca²⁺对厌氧颗粒污泥膨胀床反应器（EGSB）处理效果的影响，并采用静态实验方法考察了Ca²⁺对厌氧颗粒污泥产甲烷活性的影响。实验结果表明，进水COD为17 000 mg/L的条件下，当Ca²⁺浓度低于6 000 mg/L时，EGSB对COD去除率达93%以上；当Ca²⁺浓度高于6 000 mg/L时，COD去除率随运行时间明显下降，并在污泥中形成大量沉淀。静态实验结果表明，废水中低浓度Ca²⁺促进了厌氧颗粒污泥的产甲烷活性，但高浓度Ca²⁺明显抑制了其产甲烷活性，这是导致高Ca²⁺浓度条件下EGSB对COD去除率降低的主要原因。研究表明，颗粒污泥产甲烷活性恢复程度随Ca²⁺浓度增加而减弱。     

Laboratory simulation of biodegradation of chemicals in surface waters: closed bottle and respirometric test

Microbial degradation is the most dominant elimination mechanism of organics from the environment. For evaluation of biodegradability of pure chemicals many standardized tests are available, but no standardized procedure for assessment of biodegradability of chemicals in surface water is agreed upon. Rates of in-situ biodegradation are usually estimated in laboratory simulation where environmental factors are reproduced to some extent. The aim of our study was to compare standardised ready biodegradability assessment, test (Closed bottle test) and its modifications employing the basic agreements on test conditions to simulate biodegradation in surface water. Standard test was modified using various natural river waters to simulate the natural environment in a simplified way. The impact of different types and amounts of nutrients and microorganisms on biodegradation was confirmed. The conditions in the recipient should be examined to extrapolate the results from ready biodegradability tests to real surface water.     

常温下ABR处理低浓度废水性能及污泥特性

研究了常温(17~25℃)下4隔室厌氧折流板反应器(ABR)处理低浓度废水的运行效果及污泥特性。在水力停留时间(HRT)为24 h,进水COD浓度为1 500、1 000和500 mg/L左右时,平均COD去除率分别为94%、93%和87%。进水COD浓度保持在500 mg/L左右,将HRT降为12 h和8 h,COD的去除率仍达到83%以上。厌氧污泥性质测定结果表明,最后隔室中的污泥浓度、颗粒化程度及产甲烷活性与其他隔室相比明显较低,说明低浓度进水对最后隔室厌氧污泥的性质影响较大。颗粒污泥扫描电镜观察显示,各隔室颗粒污泥内部微生物组成差异较大,第1隔室颗粒污泥以产甲烷球菌为主,第2隔室颗粒污泥中没有明显的优势菌,但杆状菌比第1隔室明显较多,第3、4隔室颗粒污泥中以索氏甲烷丝菌为优势菌。     

Biodegradation potential of cyano-based ionic liquid anions in a culture of Cupriavidus spp. and their in vitro enzymatic hydrolysis by nitrile hydratase

Biodegradation tests with bacteria from activated sludge revealed the probable persistence of cyano-based ionic liquid anions when these leave waste water treatment plants. A possible biological treatment using bacteria capable of biodegrading similar compounds, namely cyanide and cyano-complexes, was therefore examined. With these bacteria from the genera Cupriavidus, the ionic liquid anions B(CN)₄ ^?, C(CN)₃ ^?, N(CN)₂ ^? combined with alkaline cations were tested in different growth media using ion chromatography for the examination of their primary biodegradability. However, no enhanced biodegradability of the tested cyano-based ionic liquids was observed. Therefore, an in vitro enzymatic hydrolysis test was additionally run showing that all tested ionic liquid (IL) anions can be hydrolysed to their corresponding amides by nitrile hydratase, but not by nitrilase under the experimental conditions. The biological stability of the cyano-based anions is an advantage in technological application, but the occurrence of enzymes that are able to hydrolyse the parent compound gives a new perspective on future cyano-based IL anion treatment.     

厌氧发酵系统中回流沼液的产甲烷活性研究

为确定两阶段厌氧发酵系统合理的污泥回流时段,对厌氧发酵系统排出沼液产甲烷活性进行了研究。通过对这些沼液在后储罐放置的不同天数(0~5 d)测定其不同的活性,用间歇实验方法测定最大比产甲烷活性(Um ax.CH4)作为厌氧污泥的产甲烷活性指标。结果表明,排出的沼液产甲烷活性在不同的时间段有所不同,经实验得出沼液放置第2 d时的产甲烷活性最高,从而确定该两阶段厌氧发酵系统排出污泥合理的回流时段应该是沼液进入后储罐的第2 d。进而为在线活性污泥与后储罐最佳活性时段污泥混合的污泥回流工艺提供理论依据。     

处理造纸废水的EGSB反应器内颗粒污泥特性

为了解高效厌氧反应器的颗粒污泥特性，研究了生产性EGSB反应器处理造纸废水不同高度的颗粒污泥的固体浓度、外观形态和产甲烷活性。采用甲烷势自动测试系统测试产甲烷活性，测试时间3d。结果表明，颗粒污泥的固体浓度以及溶解性COD随高度的增加逐渐降低，有机质含量沿整个反应器高度不变。反应器内固体浓度和有机质含量平均值为Ts：（123±4）g／L，VS：（75±3）g／L，VS／TS：（61±3）％。溶解性COD浓度由723mg／L降低到449mg／L。反应器内的颗粒污泥产甲烷活性为（0．32±0．03）gCOD／gVSSd（112mLCH。／gVSSd）。反应器底部和顶部颗粒污泥的当量直径分别为169μm和145μm，颗粒污泥呈椭球状（圆度为2．6）。EGSB反应器内由于进水的上升流速较高，污泥颗粒处于膨胀状态，致使颗粒污泥特性沿反应器高度变化不大。     

Ca2+对上流式多级厌氧反应器处理蔗渣渗滤液的影响

研究了上流式多级厌氧反应器(UMAR)处理蔗渣渗滤液过程中Ca2+浓度对反应器运行特性和颗粒污泥性质的影响。结果表明,对于蔗渣渗滤液而言,进水中低浓度的Ca2+浓度(80～300 mg/L)对颗粒污泥产甲烷活性无抑制,COD去除率最高可达93.3%;Ca2+浓度达到500 mg/L以上时,对厌氧颗粒污泥的活性有抑制作用;随着Ca2+浓度进一步升高,抑制作用加强,污泥灰分明显升高,活性成分下降,COD去除效率明显降低,污泥粘结沉积在反应器底部,导致系统内循环出现障碍。因此,在实际生产中应减少废水中Ca2+的引入量。     

Biodegradability of the X-ray contrast compound diatrizoic acid, identification of aerobic degradation products and effects against sewage sludge micro-organisms

Pharmaceuticals and contrast media have been detected in hospital effluents, sewage treatment plants, surface water, and ground water. Only little is known about their elimination during sewage treatment and effects of possible biotransformation products against bacteria. The modified Zahn-Wellens test (ZWT, OECD 302 B) and a test simulating biological sewage treatment (modified OECD 303 A test) were used to assess the biodegradability of the widely used ionic iodinated contrast agent diatrizoic acid (diatrizoate). Effects against sewage sludge bacteria were studied in the two test systems by monitoring the biomarkers quinones, polyamines, phospholipids and adenosine triphosphate. Diatrizoate was biotransformed into 2,4,6-triiodo-3,5-diamino-benzoic acid in the ZWT. 2,4,6-Triiodo-3,5-diamino-benzoic acid was stable under the test conditions of the ZWT. Diatrizoate was not eliminated in the OECD 303 A simulation test. It was not adsorbed by the sewage sludge. No effects of the test compound or its aerobic transformation products against the bacteria present in the sewage sludge were detected using phospholipids, quinones, polyamines, and adenosine triphosphate as biomarkers.     

Anaerobic degradation and toxicity of commercial cationic surfactants in anaerobic screening tests

Anaerobic biodegradability and toxicity on anaerobic bacteria of di(hydrogenated tallow) dimethyl ammonium chloride (DHTDMAC) and two esterquats have been investigated. A batch test system containing municipal digester solids as a source of anaerobic bacteria, based on the method proposed by the ECETOC, has been applied. To evaluate the potential toxicity of such surfactants on anaerobic sludge, a co-substrate, an easily biodegradable compound in anaerobic conditions, has been added to the samples to test and the effects on biogas production have been determined. For the esterquats studied high biodegradation levels were obtained and no toxic effects on anaerobic bacteria were observed even at the highest concentrations tested, 100 and 200 mg C/l, respectively. On the contrary, DHTDMAC was not degradated at the same test conditions. However, no inhibitory effects on the biogas production were detected for this surfactant at concentrations <100 mg C/l.     

常温下IC反应器启动过程中的颗粒污泥性能研究

在常温下用自配葡萄糖废水启动IC反应器,研究了厌氧颗粒污泥的形成过程和特性。IC反应器进水浓度为3 000 mg COD/L,水温为14.5～26℃,25 d内形成了颗粒污泥。结果表明,随着运行时间和容积负荷的增加,颗粒污泥粒径逐渐增大。反应器启动完成后,反应器中2 mm的颗粒污泥增加到6.6%,0.3 mm的颗粒从57.7%减少到39.4%;VSS浓度从24.7 g/L上升到48.2 g/L;VSS/SS从34.4%增加到72.8%。颗粒污泥的沉降速度与颗粒粒径成正比,0.3～3 mm的颗粒污泥的沉降速度介于34.05～109.75 m/h之间,具有良好的沉降性能。初始接种污泥几乎没有产甲烷活性,与第30 d的初期颗粒污泥相比,成熟的颗粒污泥的产甲烷活性提高了46.7%。     

Effect of solids concentration on bacterial leaching of heavy metals from sewage sludge

Microbial metal leaching from sewage sludge (2-9% w/v) was carried out with the iron-oxidizing bacterium Thiobacillus ferrooxidans. Measurements of pH, oxidation-reduction potential, and concentration of Fe2+ indicated that T. ferrooxidans was effective in removing metals from an incubation bath containing less than 5% sludge solids concentration. Specifically, Cu leaching was completely suppressed at a high solids concentration of 9% (w/v). Results indicated that the deactivation of T. ferrooxidans at a high sludge content was mainly due to the presence of inhibiting materials such as organic matter. A mixed culture of sulfur-oxidizing bacteria was obtained by enrichment from anaerobically digested sewage sludge to enhance the efficiency of the microbial leaching process. These bacteria were much more effective in metal leaching than was iron-oxidizing T. ferrooxidans. At 9% (w/v) solids concentration, the leaching efficiencies of Zn and Cu were 78% (2.66 g/kg dry sludge) and 59% (1.36 g/kg dry sludge), respectively. Therefore, when removing heavy metals from the anaerobically digested sewage sludge, the indigenous sulfur-oxidizing bacteria isolated in the current study were more efficient than T. ferrooxidans, especially at high sludge solids concentrations.