锦集
RMS 通过厌氧氨氧化生物抑制TN的去除性能
RMs 可以显著提高厌氧氨氧化菌关键酶的活性
RMs是推断发挥作用的Q/QH2在厌氧氨氧化过程
作为主要的原因，可能会阻止ladderane RMs 和关键酶之间的联系
文章信息
1.文章历史
2013年9月修订
2013年10月31日修订编版
2013年11月1日被接受
2013年11月10日在网上发布
2.关键词
厌氧氨氧化
氧化还原介质
肼脱氢酶
亚硝酸盐还原酶
硝酸还原酶
3.摘要
本研究shou先探讨厌氧氨氧化生物/关键酶和醌的氧化还原介质之间的活动关系，其中蒽醌-2,6-二磺酸（AQDS），2-羟基-1,4- napthoqui-无（LAW）和蒽醌-2-羧酸（AQC）。实验结果表明，总脱氮性能随三种氧化还原介质（RMS）用量的增加而呈下降趋势。例如，当AQC增加到0.8毫米，TN的去除率急剧减少到17.2mg-N/gVSS/h，只能控制大约20%。这种现象可能是微生物中毒与细胞外的RM增加而引起的。然而，粗肼脱氢酶，亚硝酸盐还原酶，和硝酸还原酶的活性增强比没有RMS的对照实验约0.6-3倍。RMS被推断在厌氧氨氧化过程中发挥辅酶/泛醌（Q/QH2）作用。此外，具体ladderane 膜结构可以阻隔RMS和厌氧氨氧化膜内的关键酶。主要原因可能是RMS对厌氧氨氧化生物和关键酶的反向影响。
1.简介
厌氧氨氧化（ANAMMOX）现在被确认为是一种新颖的重要的生物脱氮工艺。它可以在厌氧条件下将NH4与NO2直接转化成N2（Strouset等人，1999）。与传统工艺相比（硝化反硝化生物），厌氧氨氧化过程提供了显著的优点，如对氧气和有机碳，低污泥产量和减少CO2和NO2的排放（Opden Campet等人，2006）。近日，唐等人（2010）报告了一个高达74.3-76.7 kg-N/m3/d的脱氮率在一个实验室规模的厌氧氨氧化UASB反应器，在废水生物脱氮的厌氧氨氧化工艺的高电位。然而，如此高的脱氮率（NRR）是通过连续添加厌氧氨氧化污泥到目标反应器，其中生物量浓度的增加高达42-57.7VSS/L（唐等人，2010）。此外，厌氧氨氧化菌相对长的培养时间为也会导致较长的启动时间，通过降低厌氧氨氧化菌的丰度使厌氧氨氧化系统更加脆弱。因此，提高生物质厌氧氨氧化菌活性，并进一步缩短厌氧氨氧反应器的启动是很趣味性和挑战性的课题。研究人员已经进行了大量的努力， 通过外场能量（磁场力，低强度超声）或添加几种微量营养元素增加厌氧氨氧化菌的活性。例如，刘等。（2008）施加的磁场成功地提高厌氧氨氧化菌的活性，在60T的磁化率下，**大脱氮率提高了30%。类似地，段等，（2011）表明，总氮（TN）厌氧氨氧化菌的去除率提高25.5%，通过施加0.3W/cm2超声强度与4分钟的**佳照射时间，这个效果可以持续6天左右。除了外部*域的应用，乔等（2012）表明Mno2粉末的加入也可以使厌氧氨氧化菌的脱氮率为无二氧化锰粉的2倍。
近日，氧化还原介质（RMS）被发现在有机和无机污染物的厌氧生物转化中起着重要的作用（范德齐和塞万提斯，2009）。有一些研究集中在氧化还原介质的反硝酸化脱氮工艺中的作用。阿兰达泰马瑞等人（2007）通过反硝化生物，包括蒽醌-2,6-二磺酸（AQDS），1,2-萘醌-4-磺酸酯e(NQS).2,6-disulfonate(AQDS), 2-羟基-1,4-萘醌，研究了同时转换硫化物和硝酸盐不同醌的氧化还原介质的影响。他们证明，NQS必须使用硫化氢作为电子供体的**高硝酸盐还原率（荷兰达泰马瑞等人，2007）。Guoet等人（2010）探讨了氧化还原介质催化脱氮工艺与蒽醌(AQ)由海藻酸钙固定。他们还发现，加入500个蒽醌固定化珠会加速脱氮约两倍。刘等（2012）证明，固定到功能聚合生物载体2-磺酸蒽醌（00.4mmol/L），可以提高脱氮率约1.5倍。直到现在还没有关于RMS对厌氧氨氧化生物有影响的报告。
反硝化生物量**关键酶位于细胞膜或细胞膜外介质。因此，RMS可以接触这些酶和加快硝酸盐或亚硝酸盐的生物降解速率。然而，厌氧氨氧化菌的关键酶是位于厌氧氨氧化菌膜内，并在其膜引起质子动力势和随后的由膜结合ATP酶合成ATP（图1中示出）。从厌氧氨氧化菌外面进入厌氧氨氧化膜，RMS必须穿过细胞壁，细胞质膜，卵胞浆内膜和厌氧氨氧化膜以便与关键酶接触。厌氧氨氧化菌膜结构由C18和C20脂肪酸组成，包括3个或5个线性级链环丁烷（sinningheet等人，2002）。它们被脂结合到甘油骨架或醚结合的烷基链（sinningheet等人，2005）。因此，ladderane可能会阻止RMS和厌氧氨氧化膜内部的关键酶之间的接触。
本研究的目的是讨调查三种RMS对厌氧氨氧化生物活性的影响。厌氧氨氧化菌的关键酶（肼脱氢酶硝酸还原酶、亚硝酸还原酶）上RMS的影响也进行了研究。还讨论了在两个厌氧氨氧化生物和关键酶上的效果的可能机制。所测试的RMS包括蒽醌-2,6-二磺酸（AQDS），2-羟基-1,4-萘醌（法）和蒽醌-2-羧酸（AQC）。
2.方法
2.1. 微生物与资料媒体
厌氧氨氧化污泥用于接种源自形反应器的厌氧氨氧化实验规模的实验室。反应器的内径和高度分别为8和45厘米。这种反应器在670天的操作的总脱氮（TN）率为 8.0 kg-N/m3/d 。 KSU-1株(AB057453.1)的厌氧氨氧化菌约占种子的总生物量的70–75%。在实验中使用的介质主要是由在（NH4）2SO4和亚硝酸钠中的铵和亚硝酸盐的形式。该微量矿物质培养基的组成如范德格拉夫等人描述（1996）。#p#分页标题#e#
2.2 批量测试
为了确定不同的RMS浓度对特殊厌氧氨氧化活性的影响，制定了七组是RMS从0到0.8毫米不同浓度的实验。该实验是在七个120毫升含100毫升培养基瓶的血清瓶，每个包含厌氧氨氧化的生物质具有不同的RMS（MLVSS浓度2000 mg/L）。生物样品取自反应器并在矿物质中洗涤三次，以除去残留的氮气。将pH值调整**7.5，将温度保持在35± 1 °C左右的水域摇床。摇动转速设定为150转的转速，保持生物量和媒体之间的充分接触。血清瓶内容物与二氮的气体被洗清以除去溶解的氧。初始NH4 -N和NO2 -N浓度都设定在50毫克/升。特别厌氧氨氧化活性，随着时间流逝由在瓶里的氨和亚硝酸的浓度每单位生物量降低浓度曲线峰值改变。使用无菌注射器每小时收集一次样品，并通过0.45lm孔径的膜吹扫来分析NH4 -N，NO2- N，NO3- N，和RMS浓度。
2.3分析方法
Concentrations of nitrite and nitrate were determined by using on-exchange chromatography (ICS-1100, DIONEX, AR, USA) with an Ion Pac AS18 anion column after ltration with 0.22 lm poresize membranes. NH4-N, MLSS and MLVSS concentrations were measured according to the Standard Methods (APHA, 1995).
硝酸盐和亚硝酸盐的浓度通过使用离子交换色谱测定法（ics-1100，Dionex，AR，USA）与0.22ml 孔径膜过滤PAC AS18阴离子柱。NH4-N，MLSS和MLVSS浓度按标准方法测定（APHA，1995）。
pH值的测量是通过使用一个数字
ph计（phs-25，雷磁公司，中G），而DO使用数字DO做计进行测量（YSI，模型55，美G）。使用分光AQDS，AQC和规律浓度均在其**大吸光度采用分光光度法测量（k = 328，336，452 nm）用分光光度计（v-560uv /VIS分光光度计，日本）根据哈藤巴赫等人。（2008）和劳等人。（2002）
2.4生物质提取物的制备及酶活性的测定
从我们的实验室规模的反应器中量取5克（湿重）厌氧氨氧化生物质。生物样品离心8000 rpm离心转，在4°C进行 20 分钟，随后用磷酸钠缓冲溶液洗涤两次（20毫米，pH 7.0）。洗涤后的沉淀在20毫升缓冲液溶解再沉淀后超声（225 W，4°C 30分钟，超声波处理器CPX 750，美G）。细胞团通过离心（22000转）分离，在4°C进行 30分钟。将上清液储存在4°C作为蛋白质和酶活性测定细胞提取物。蛋白质浓度的测量根据布拉德福德（布拉德福德法，1976），以牛血清白蛋白为标准。据岛村等人描述的肼脱氢酶酶活性方法测定（2007），并且反应用分光光度计描绘为在550 纳米处增加在标注混合物中细胞色素C的吸光度（v-560紫外可见分光光度计，雅有限公司，日本）。该混合物由100毫米磷酸钾缓冲液（pH 7.0），50lm马心脏细胞色素C（氧化型），酶解液适量，和25lm肼。肼脱氢酶（HDH）活性表示为细胞色素减少／毫克蛋白/分钟。硝酸还原酶（NAR）的活性测定Meincke等人（1992）通过测定亚硝酸盐消耗方法。氯酸钠作为电子受体。测定含有22 mm NaClO3和11毫米50毫米亚硝酸钠钾磷酸盐缓冲液，pH值7。反应通过加入酶开始。一个单位的酶活性被denedaslmol 亚硝酸盐还原酶（NIR）的活性作为赛义德在Hira等人描述的方法的基础上（2012）以减少甲基紫（MV）作为电子供体。反应混合物含有100 毫米磷酸钾缓冲液（pH7.0），MV（3毫米），亚硝酸钠（6毫米）和0.1毫升的生物质提取物在塞紧的4毫升试管内制备。反应是由连二亚硫酸钠的注射开始（12毫米）。酶活性被作为亚硝酸盐的还原。所有的测定混合物要在35± 1°C进行。
2.5系统分析
所有在本文中提出的数据是一式三份，实验的数据的平均值。通过使用单因素方差分析每与邓肯的多范围检验（SPSS 19），和每次形成统计分析的P＜0.05值被认为是统计学显著性。
英语原文
Effects of quinoid redox mediators on the activity of anammox biomass
highlights
RMs addition depressed TN removal performance by anammox biomass.
RMs could markedly enhance the key enzymes activities of anammox bacteria.
RMs was inferred to play the role as Q/QH2 during anammox process.
Ladderane as the main reason might block the contact between RMs and key enzymes.
Article info
Article history:
Received 19 September 2013
Received in revised form 31 October 2013
Accepted 1 November 2013
Available online 10 November
2013
Keywords:
Anammox
Redox mediator
Hydrazine dehydrogenase
Nitrite reductase
Nitrate reductase
abstract
This study rst explored the relationship between the activity of anammox biomass/key enzymes and quinoid redox mediators, which were anthraquinone-2,6-
disulfonate (AQDS),2-hydroxy-1,4-napthoqui-none (LAW) and anthraquinone-2-carboxylic acid (AQC). Experimental results demonstrated that the total nitrogen removal performance showed a downward trend with all three redox mediators (RMs) dosage increasing. For instance, when the AQC addition increased to 0.8 mM, the TN removal rate sharply reduced to 17.2 mg-N/gVSS/h, only about 20% of the control. This phenomenon might be caused by microbial poisoning with the extracellular RMs additions. Nevertheless, the crude hydrazine dehydroge-nase, nitrite reductase, and nitrate reductase activities were enhanced with RMs addition, about 0.6–3folds compared to the control experiments without RMs addition. The RMs was inferred to play the role as ubiquinol/ubiquinone (Q/QH2) during the anammox process. Furthermore, the specic ladderane membrane structure could block the contacting between RMs and the key enzymes inside anammox-some. This might be the main reason for the contrary effects of RMs on anammox biomass and the key enzymes.#p#分页标题#e#
1. Introduction
Anaerobic ammonium oxidation (anammox) process is now
recognized as a novel and important process in biological nitrogen removal, which can directly convert NO 2 to N2 gas with NH4 under anaerobic conditions (Strous et al., 1999). Compared with the conventional biological processes (nitrication–denitrication),anammox process offers signicant advantages such as no demand for oxygen and organic carbon, low sludge production and reduced
CO2 or N2O emissions (Opden Campet al., 2006).Recently, Tang
et al. (2010) reported a very high nitrogen removal rate of 74.3–76.7 kg-N/m3/d in a lab-scale anammox UASB reactor, which demonstrated high potential of anammox process in biological nitrogen removal from wastewaters. However, such a high nitrogen removalrate (NRR) was achieved through the continuous addition of anammox seed sludge into the targeted reactor, in which the biomass
concentration increased as high as 42.0–57.7 g-VSS/L (Tanget al.,2010). Furthermore, the relative long doubling time of anammox bacteria will also cause a longer startup period and make the anammox system more vulnerable with low anammox bacteria abundance. Consequently, enhancing the bacterial activity of anammox biomass and further shortening the start-up period of anammox reactors are subjects of great interest and challenge.
Researchers have made numerous efforts to increase the activity of anammox biomass by utilizing external eld energy (mag-netic eld, low intensity ultrasound) or adding some kinds of micronutrient. For instance, Liu et al. (2008) applied magnetic eld successfully to enhance the activity of anammox bacteria whereby the maximum nitrogen removal rate increased by 30% at magnetic value of 60.0 mT in long term. Similarly, Duan et al. (2011) demon-strated that total nitrogen (TN) removal rate of anammox bacteria increased by 25.5% by applying ultrasound intensity of 0.3 W/cm2 with the optimal irradiation time of 4 min, and this effect could last
for about 6 days. Besides the application of external eld, Qiaoet al. (2012) demonstrated that the addition of MnO2 powder could also increase the nitrogen removal rate of anammox biomass about 2 times as high as that without MnO2 powder addition.
Recently, redox mediators (RMs) were found to play an important role in the anaerobic transformation of organic and inorganic contaminants (Van der Zee and Cervantes, 2009).There were a few studies focused on the role of redox mediators on nitrogen removal by denitrication process. Aranda-Tamaura et al.(2007) investi-gated the impacts of different quinoid redox mediators on the
simultaneous conversion of sulphide and nitrate by denitrifying biomass, including anthraquinone-2,6-
disulfonate (AQDS), 2-hy-droxy-1,4-naphthoquinone and 1,2-naphthoquinone-4-sulphonate(NQS).They demonstrated that NQS had the highest nitrate reduc-
tion rate using sulphide as electron donor (Aranda-
Tamaura et al.2007). Guo et al. (2010) explored the possibility of redox mediator catalyzing denitrication process with anthraquinone (AQ) immo-bilized by calcium alginate. They also found that addition of 500 anthraquinone immobilization beads would accelerate the denitri-fying rate about 2 times. Liu et al. (2012) demonstrated that anthraquinone-2-sulfonate (0.04 mmol/L) immobilized into the functional electropolymerization biocarriers could increase the
denitrication rate about 1.5 folds. Until now there was no report on the effects of RMs on anammox biomass.
Most key enzymes of denitrifying biomass are located on the cell membrane or the cell membrane periplasma. Thus, RMs could contact these enzymes and accelerate the biodegradation rate of nitrate or nitrite. However, all the key enzymes of anammox bac-teria are located inside anammoxosome, and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by
Membrane-bound ATPases (shown in Fig.1). From the outside of anammox bacteria into anammoxosome, RMs must cross cell wall,cytoplasmic membrane, intracytoplasmic membrane and anammox some membrane in order to contact with the key enzymes.
The ladderane of the anammoxosome membrane consist of C18
and C20 fatty acids including either 3 or 5 linearly concatenated cyclobutane rings (Sinninghe et al., 2002). They are ester bound to a glycerol backbone or ether bound as alkyl chains (Sinninghe et al., 2005). Therefore, the ladderane might block the contacting between RMs and the key enzymes inside anammoxosome.
The objective of this study was to investigate the effects of three kinds of RMs on the activity of anammox biomass. The effects of RMs on the key enzymes (hydrazine dehydrogenase nitrate reductase and nitrite reductase) of anammox bacteria were also studied.The possible mechanisms of effects on both anammox biomass and the key enzymes were also discussed. The tested RMs included anthraquinone-2,6-disulfonate (AQDS), 2-hydroxy-1,4
-naphtho-quinone (LAW) and anthraquinone-2-carboxylic acid (AQC).
2. Methods
2.1. Microorganisms and feed media
The anammox sludge used for inoculation originated from a laboratory-scale anammox upow column reactor in our lab. The inner diameter and height of the column-type reactor were 8 and 45 cm, respectively. The working volume of this reactor was 2 Land continuously operated under 35 ± 1 °C. The total nitrogen(TN) removal rate of this reactor reached 8.0 kg-N/m3/d during 670 days’operation.
Anammox bacteria of KSU-1 strain(AB057453.1) accounted for about 70–75% of the total biomass in seed biomass. The media used in the experiments mainly consisted of ammonium and nitrite in the form of (NH4)2SO4 and NaNO2. The composition of the trace mineral medium was as described by vander Graaf et al. (1996).#p#分页标题#e#
2.2. Batch experiments
In order to ascertain the effects of different RMs concentrations on specic anammox activity, seven sets of batch experiments were conducted with the RM concentration from 0 to 0.8 mM.The tests were carried out in seven 120 ml serum vials containing 100 ml medium, each containing anammox biomass (MLVSS con-centration of 2000 mg/L) with varied RMs additions. Biomass sam-ples were taken from the reactors and washed three times with
mineral medium to remove residual nitrogen. The pH was adjusted to 7.5 and the temperature was maintained at 35 ± 1 °C in a water bath shaker. The shaking speed was set at 150 rpm to keep the full contact between biomass and media. The serum bottle contents were purged with dinitrogen gas to remove dissolved oxygen. Ini-tial NH
4 -N and NO2 -N concentrations were set at 50 mg-N/L. Spe-
cic anammox activity was estimated from the peak of the curve indicated by the decrease of ammonium and nitrite concentrations per unit biomass concentration in the vials as time lapsed. The samples were collected every hour using a sterile syringe and purged through 0.45
lm pore size membranes to analyze the NH4 -N, NO2 -N, NO3 -N and RMs concentrations.
2.3. Analytical methods
Concentrations of nitrite and nitrate were determined by using ion-exchange chromatography (ICS-1100, DIONEX, AR, USA) with an IonPac AS18 anion column after ltration with 0.22lm pore size membranes. NH4-N, MLSS and MLVSS concentrations were measured according to the Standard Methods (APHA, 1995). pH measurement was done using a digital pH meter (PHS-25, Leici Company, China), while DO was measured using a digital DO meter(YSI, Model 55, USA). The concentrations of AQDS, AQC and LAW were measured spectrophotometrically at their absorbance maxi-mum (k = 328, 336 and 452 nm, respectively) using a spectropho-
tometer(V-560UV/VIS Spectrophotometer,Jasco,Japan) according to Hartenbach et al. (2008) and Rau et al. (2002).
2.4. Preparation of biomass extracts and determination of enzyme activity
5 g (wet weight) anammox biomass was taken from our lab-scale reactor. The biomass samples were centrifuged at 8000 rpm at 4 掳C for 20 min followed by washing twice with sodium phos-phate buffer solution (20 mM, pH 7.0). The washed pellets were then resuspended in 20 ml of the same buffer and lysed by freezing and thawing followed by sonication (225 W, at 4 掳C for 30 min,
Ultrasonic processor CPX 750, USA). Cell mass was separated by centrifugation (22 000 rpm), at 4 掳C for 30 min. The supernatant was stored at 4 掳C and used as cell extract in the determination of protein and enzyme activity. Protein concentration was measured according to the Bradford procedure (Bradford, 1976), using BSA
as a standard. Enzyme activity of hydrazine dehydrogenase was according to the methods described by Shimamura
et al. (2007), and the reactions were depicted as an increase in the absorbance of cytochrome c at 550 nm in the standard mixture using a spectrophotometer (V-560 UV/VIS Spectrophotometer, Jas-co, Japan). The mixture consisted of 100 mM potassium phosphate buffer (pH 7.0), 50lM horse heart cytochrome c (oxidized form),an appropriate amount of enzyme solution, and 25 lM hydrazine.The hydrazine dehydrogenase (HDH) activity was expressed as
lmol of cytochrome c reduced/mg protein/min. Nitrate reductase
(Nar) activity was assayed in accordance with the methods re-corded by Meincke et al. (1992) by measuring the nitrite consump-tion. NaClO3 was used as electron acceptor. Assays contained 22 mM NaClO3 and 11 mM NaNO2 in 50 mM potassium phosphate buffer, pH 7.0. The reaction was started by the addition of the en-zyme. One unit of enzyme activity was dened as lmol of nitrite oxidized/mg protein/min. Nitrite reductase (Nir) activity was as-sayed on the basis of the methods described by Hira et al. (2012)using reduced methyl viologen (MV) as electron donors. The reac-tion mixture containing 100 mM potassium phosphate buffer (pH
7.0), MV (3 mM), sodium nitrite (6 mM) and 0.1 ml biomass extracts was anaerobically prepared in a stoppered 4.0 ml cuvette. The reac-tion was started by the injection of sodium dithionite (12 mM). Aunit of enzyme activity was dened as lmol of nitrite reduced/mg protein/min. All the assay mixtures were incubated at 35 卤 1 掳C
2.5. Statistical analysis
All the data presented in this paper were the mean values of data from triplicate experiments. Statistical analysis was per-formed by using one-way ANOVA with the Duncan’s multiple range test (SPSS 19.0) and values of p < 0.05 were considered to be statistically signicant.
