系统分析臭氧处理过的污泥的微生物生化性能和生物群落的变化

Abstract:Two lab-scale bioreactors (reactors 1 and 2) were employed to examine the changes in biological performance and the microbial community of an activated sludge process fed with ozonated sludge for sludge reduction. During the 122 d operation, the microbial activities and community in the two reactors were evaluated. The results indicated that, when compared with the conventional reactor (reactor 1), the reactor that was fed with the ozonated sludge (reactor 2) showed good removal of COD, TN and cell debris, without formation of any excess sludge. In addition, the protease activity and intracellular ATP concentration of reactor 2 were increased when compared to reactor 1, indicating that reactor 2 had a better ability to digest proteins and cell debris. DGGE analysis revealed that the bacterial communities in the two reactors were different, and that the dissimilarity of the bacterial population was nearly 40%. Reactor 2 also contained more protozoa and metazoa, which could graze on the ozone-treated sludge debris directly.摘要：利用两个实验室规模的生物反应器（反应器1和2)加以检验活性污泥工艺运用污泥臭氧氧化分解法的与否，生物学性能和生物群落的变化及活性污泥法处理污泥减少的效果。在122天的操作过程中,对两个反应器在微生物活动和群落进行评估。结果表明，与常规反应器（1)相比,输送臭氧氧化污泥的反应器（2）表现出了更好的COD、TN和细胞碎片去除效果,并且没有形成任何多余的污泥。此外, 反应器2较反应器1中蛋白酶活性和细胞内 ATP浓度增加更多，且反应器2具有更好的消化蛋白质和细胞碎片的能力。DGGE分析显示两个反应器不同细菌群落数接近40%。反应器2又含有较多的原生动物、后生动物,能直接吃掉经过臭氧处理过的污泥的碎片。1. Introduction:The use of activated sludge as a biological wastewater treatment process has been employed to treat a wide variety of wastewater.However, its primary by-product, excess sludge, has become a large problem. Indeed, treatment and disposal of excess sludge can account for up to 60% of the total operational costs of a wastewater treatment plant (Spellman, 1997). For this reason, it is important to develop methods of reducing excess sludge produced during wastewater treatment in an economic, environmentally safe and practical manner. One promising technique is the sludge ozonation process (Yasui et al., 1996; Wei et al., 2003; Yan et al.,2009). Ozone is a strong chemical oxidant capable of destroying the cell walls of microorganisms and solubilizing or oxidizing them to organic substances.1.论文简介:利用活性污泥作为生物污水处理的工艺已被用来治理各式各样的废水。然而,它的主要副产品剩余污泥,已经成为一个大问题。事实上,剩余污泥的处理处置费用可以占总运营成本的60%(Spellman污水处理厂,1997)。基于这个原因,以减少剩余污泥为目的在废水处理过程中产生的一个经济的、环保的和务实的态度成为重要的发展方式。当前运用污泥臭氧氧化分解法等过程是一个有前景的技术(Yasui et al., 1996; Wei et al., 2003; Yan et al.,2009)。臭氧是一种强化学氧化剂可以摧毁细胞壁内含有微生物并使其增容、氧化它们有机物质。The sludge ozonation process has been defined as a sequence of decomposition processes that includes disintegration of suspended solids, solubilization of the solids (cells) and mineralization of the soluble organic matter released from the microbial cells (Ahn et al., 2002). Recently, we comprehensively studied the mechanism of sludge ozonation at different ozone dosages using a combination of biological and chemical approaches (Yan et al., 2009), including analysis of the changes in biological response based on changes in the CFU and PCRDGGE profiles, bio-macromolecular activity and radical-scavenging activity. The results revealed that, when the ozone dosage was as high as 0.14 to 0.27 g O3/g TSS, ozone failed to oxidize the sludge matrix efficiently due to the release of radical scavengers such as lactic acid and from the microbial cells in the sludge. These findings indicate 2SO4that prolongation of the sludge ozonation process causes the ozone to gradually lose its ability to oxidize sludge solids and soluble organic molecules. In addition,these findings indicated that the efficiency of sludge decomposition by ozonation has its limits, even at high ozone doses. The sludge ozonation process has been employed to reduce excess sludge by feeding ozonated sludge into the activated sludge (Yasui and Shibata, 1994; Ahn et al., 2002; Bohler and Siegrist, 2004; Cui and Jahng, 2004). During biological treatment, a portion of the recirculated ozone-treated sludge is oxidized to CO2 in the aeration tank, while another portion is turned into new microbial cells. This process is known as the lysis-cryptic growth process, which is considered to be an important mechanism for the reduction of sludge by sludge ozonation (Wei et al., 2003).污泥臭氧氧化过程已经被定义为一种序列的热分解过程,包括分解悬浮的固体颗粒,使固体颗粒(细胞)增容，矿化可溶的有机质和释放微生物细胞等(Ahn et al., 2002)。最近,我们进行了臭氧结和生物和化学方法在不同剂量下综合研究污泥臭氧氧化分解法的机理 (Yan et al., 2009),包括分析菌落数、多聚酶链反应-变性梯度凝胶电泳配置文件、大分子生物活动和原子团清除活动变化的基础上分析生物反应的变化。结果显示,当臭氧剂量高达0.140.27gO 3/g TSS时，由于自由基清除剂的释放，例如来自污泥中微生物细胞的乳酸和 ，2SO4使臭氧未能有效的氧化污泥基质。这些发现表明,随着污泥臭氧氧化过程的延长,导致臭氧逐渐失去氧化污泥固体和使有机分子溶解的能力。此外,这些研究结果表明,即使是在很高的臭氧剂量下，污泥臭氧氧化分解法的效率有其限制。通过将臭氧氧化污泥输送到活性污泥中，污泥臭氧氧化过程已经被用来减少剩余污泥(Yasui and Shibata, 1994; Ahn et al., 2002; Bohler and Siegrist, 2004; Cui and Jahng, 2004)。在生物处理过程中 ,循环臭氧氧化污泥一部分在曝气池被氧化成CO 2,另一部分变成了新的微生物细胞。这个过程被称为溶胞隐性生长过程,并被认为是通过污泥臭氧氧化过程减少污泥的一个重要机理(Wei et al., 2003)。Many studies have evaluated the effects of introducing ozonated excess sludge into a variety of activated sludge reactors, including traditional activated sludge reactors (Yasui and Shibata, 1994;Yasui et al., 1996), AO reactors (Cui and Jahng, 2004; Saktaywin et al., 2005), sequenced batch reactors (SBR) (Huysmans et al.,2001) and membrane bioreactors (MBRs) (Oh et al., 2007). In these studies, a fraction of the recycled sludge passing through the reactor is always treated by ozonation, and the ozonated sludge is then fed back to the aeration tank for biological treatment together with the wastewater. Although ozonation-assisted sludge reduction processes have been successfully developed in practice,there are still several problems that should be considered. For example, the sludge system and wastewater conditions often differ among different experiments reported in previous studies; therefore,comparison of activated sludge employing sludge ozonation is difficult. Furthermore, several studies have reported that increases in the amount of inorganic solid may occur in aeration tanks and effluent water quality may be dependent on the operating conditions when the ozone-treated sludge was fed into the aeration tank (Yasui and Shibata, 1994; Yasui et al., 1996; Bohler and Siegrist, 2004). However, most previously conducted studies have focused on the operating conditions and the performance of the activated sludge process at the time of introduction of the ozonated sludge (Yasui and Shibata, 1994; Yasui et al., 1996; Sakai et al.,1997; Ahn et al., 2002; Cui and Jahng, 2004; Lee et al., 2005; Saktaywin et al., 2005). After the introduction of ozonation to the activated sludge processes, nutrients released by cell lysis and cell debris may alter the influent characteristics of the entire system;however, there have been few in depth analyses of the biological performance and microbial communities associated with the activated sludge process when ozonated sludge was introduced to reduce the sludge.许多研究评价了在各种活性污泥工艺中臭氧氧化剩余污泥的效果,包括传统的活性污泥工艺(Yasui and Shibata, 1994;Yasui et al., 1996),AO反应器(Cui and Jahng, 2004; Saktaywin et al., 2005), 序批式活性污泥法 (SBR) (Huysmans et al.,2001)和膜生物反应器(MBRs) (Oh et al., 2007)。在这些研究中,总是回收利用一小部分污泥通过臭氧氧化反应器处理,然后将臭氧氧化污泥反馈到曝气池连同废水一起生物处理。虽然辅助臭氧以减少污泥的过程已成功地在实践中发展,仍有几个应该考虑进行考虑的问题。例如, 在过去的研究中不同的实验报告污泥系统及污水条件往往是不同的,因此比较采用活性污泥使用污泥臭氧氧化法是很困难的。此外，一些研究报告表明当臭氧处理过的污泥是反馈进入曝气池时，曝气池中的无机固体量可能增加且出水水质可能依赖于运行条件(Yasui and Shibata, 1994; Yasui et al., 1996; Bohler and Siegrist, 2004)。然而，在引进污泥臭氧氧化时，大多数以前进行的研究都集中在工作条件和活性污泥法处理的性能(Yasui and Shibata, 1994; Yasui et al., 1996; Sakai et al.,1997; Ahn et al., 2002; Cui and Jahng, 2004; Lee et al., 2005; Saktaywin et al., 2005)。但引进臭氧氧化污泥对活性污泥进行减产的过程后，细胞裂解液和细胞碎片释放的营养物质，可能会改变整个系统的进水特性;然而，仅在深入分析的生物性能和活性污泥的微生物群落等有数过程时，臭氧氧化污泥已经运用于减少污泥。The sludge matrix includes bacteria, protozoa and metazoans,which all contribute to the function of the activated sludge in the aeration tank. Furthermore, each of the microorganisms has different growth patterns, growth rates and dependence on the environmental conditions. After the ozone-treated sludge consisting of the cell debris and soluble organics released from the disrupted cells(Yan et al., 2009) is returned to the bioreactor for degradation,the influent of the bioreactor will be altered. Indeed, the presence of a large amount of cell debris and soluble organics in the influent leads to cryptic growth (Wei et al., 2003) when the ozonated sludge solution is returned to the bioreactor, which may influence all of the microbes in the bioreactor. Lysis-cryptic growth may be induced in one of two ways. The first possibility is that the bacteria in the sludge may secrete hydrolysis enzymes that help hydrolyze the debris. This causes changes in the bacterial hydrolysis activity and succession in the bacterial population (Mason et al., 1986). The second way that lysis-cryptic growth may be induced is through the direct consumption of cell debris by protozoa and metazoans in the activated sludge, which leads to quick propagation of the microfauna.污泥基质包括细菌，原生动物和后生动物，这一切有助于在曝气池活性污泥的功能。此外，每个微生物的都有不同的增长模式，增长速度和环境条件的依赖。臭氧处理后的污泥组成的细胞碎片，从中释放出来的可溶性有机物(Yan et al., 2009)返回到生物反应器进一步降解，以至于生物反应器的进水水质将被改变。事实上，大量的细胞碎片和可溶性有机物存在于进水时将导致活性污泥神秘的增长(Wei et al., 2003)，当臭氧氧化污泥的解决方案返回到反应器，这可能会影响生物反应器中所有的微生物。溶胞隐性生长过程可能是两种情况之一的诱因。第一种可能性是，在污泥中的细菌可能分泌有助于细胞碎片水解的水解酶。这会导致细菌的水解活性和细菌群体的后代的变化(Mason et al., 1986)。溶胞隐性生长过程可能诱发的第二种方式是通过活性污泥中的原生动物和后生动物直接消耗细胞碎片，从而导致活性污泥中的微生物迅速繁殖。In this study, two lab-scale bioreactors (reactor 1 and reactor 2)were employed to systematically examine changes in the biological performance and microbial community structure of an activated sludge process that was fed with ozonated sludge to realize sludge reduction. Reactor 1, which was used as a control,was a conventional activated sludge reactor from which the excess sludge was withdrawn periodically. Reactor 2 contained the same activated sludge as reactor 1, but this sludge was combined with a batch sludge ozonation process in which the excess sludge that was withdrawn was subjected to ozonation. The excess sludge withdrawn from reactor 2 was treated by ozonation, after which it was continuously fed to reactor 2. The sludge activities such as the protease activity, intracellular ATP concentration and levels of anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CA) were measured during the continuous operation. The bacterial population was analyzed by PCRDGGE, and the microfauna structure in the two reactors was evaluated by microscopic observation. Taken together, the results of this study can provide a comprehensive basis for practical application of an ozone-based sludge reduction system.在这项研究中，两个实验室规模的生物反应器（反应器1和反应器2）系统地研究了在一种活性污泥工艺中是否输入臭氧氧化污泥与否的生物性能和生物群落结构的变化，及实现污泥减产的效果。 反应器 1用来作为对照，是一个传统活性污泥法反应器，定期清理剩余污泥。反应器2采用相同的活性污泥法，但反应器2中定期清理出的剩余污泥要经过臭氧氧化处理。从反应器2清出的剩余污泥经臭氧处理后，它源源不断地输送回反应器2。通过测定蛋白酶活性反应污泥活动，在系统连续运行时分别测定细胞内ATP含量和水平抗氧化酶素，如超氧化物歧化酶（SOD）和过氧化氢酶（CA）。采用PCR - DGGE技术分析菌群数目，使用显微镜观察评估两座反应器内的微生物结构。两者合计，这项研究的结果可以为臭氧化污泥减量化系统的实际应用提供一个综合性的基础。2. Methods2.1. Lab-scale activated sludge reactors 1 and 2Two lab-scale simulated activated sludge reactors (1 and 2) were established (Table 1). Each reactor contained a 4.6 L bioreactor and a 5.9 L settling tank. For start-up of the two reactors, the returned sludge of a municipal wastewater treatment plant (Qinghe, Beijing, China) was inoculated and cultivated with synthetic wastewater (Feng et al., 2008) to domesticate the two reactors for about 30 d. The primary components of the synthetic wastewater were glucose (chemical oxygen demand (COD) concentration of about 800 mg/L), (NH4)2SO4 (total nitrogen (TN) concentration of about 40 mg/L), KH2PO4 (total phosphorus (TP) concentration of about 5 mg/L), MgSO4 (9.5 mg/L), CaCl2 (0.91 mg/L) and Fe2(SO4)3 (0.14 mg/L). After 30 d of domestication, reactor 1 was operated as a traditional activated sludge reactor, with 300 mL of excess sludge being withdrawn from the bioreactor once a day.Reactor 2 contained a batch ozonation unit that was used to ozonate 300 mL of the excess sludge that was discharged from the reactor at a dose of 0.15 g O3/g MLSS using a ozone generator(ED-OG-R4, Eco Design, Japan) (Yan et al., 2009). The ozonated sludge was continuously returned to the bioreactor together with the synthetic wastewater at a ratio of 0.3:8 (v/v). During the operation in reactor 2, no sludge was discharged from the reactor. Following treatment with 0.15 g O3/g MLSS ozone, approximately 70% of the mixed liquor suspended solids (MLSS) was reduced and approximately 1000 mg/L soluble chemical oxygen demand(SCOD), 100 mg/L TN and 18 mg/L TP were released from the ozonated sludge. Table S1 (Supplementary data) shows the MLSS,SCOD, TN and TP of the sludge before and after ozone treatment. The results revealed that MLSS reduction rate of as high as 73.5% was obtained, indicating that the sludge in the lab was more easily oxidized when compared with the practical sludge taken from the wastewater treatment plant (Yan et al., 2009). These differences might be caused by the sludge in the lab being cultivated using synthetic wastewater.The average influent COD, TN and TP are shown in Table 1. The HPLC method was used to determine the level of organic acid in the influent. The results of HPLC showed that the influent of reactor 2 contained more organic acid than that of reactor 1, and that these organic acids were released by the ozonation of the sludge (data not shown). The solid residence time (SRT) of the activated sludge was approximately 15 d, and the hydraulic retention time (HRT) of the artificial sewage was 10 h. The dissolved oxygen (DO) was controlled at 3 mg O2/L in the two bioreactors and below 0.1 mg O2/L in the settling tanks. The internal recycling rate (R) of the sludge from the settling tank to the bioreactor was controlled at about0.75 (v/v). To reach a stable state for an extended period, the total running time of the two different reactors was greater than 120 d without temperature control (from April to August in 2008). The temperature of the reactors changed from 21 to 30, with an increase from 22 to 27occurring from the first to the 50th day (from May 26) and the temperature being maintained at approximately 29after the 60th day (Fig. S1).2方法2.1.实验室规模的活性污泥反应器1和2 建立了两个实验室规模模拟活性污泥反应器（1和2）（见表1）。每个反应器包含一个4.6升的生物池和5.9升的沉淀池。两个反应器的开始阶段使用同一个城市污水处理厂的回流污泥 (Qinghe, Beijing, China)接种到合成废水(Feng et al., 2008)中驯化30天。合成废水的主要成分是葡萄糖（化学需氧量（COD）约800mg/L的浓度），（NH 4） 2SO4（总氮（TN）的浓度约为40mg/L），磷酸二氢钾（总磷（TP ）浓度约5mg/L），硫酸镁（9.5mg/L ），氯化钙（0.91mg/L）和Fe2（SO 4） 3（0.14mg/L）。经过30天的驯化后，反应器1作为一种传统的活性污泥反应器，每天从生物反应却内排除300mL剩余污泥。反应器2配有一批单位臭氧化装置，将从反应器2中排出300mg剩余污泥在0.15 g O3/g MLSS的剂量使用臭氧发生器(ED-OG-R4, Eco Design, Japan)臭氧氧化(Yan et al., 2009)。经过臭氧氧化的污泥以0.3:8 的体积比不断返回到生物反应器与合成废水混合到一起。在反应器运行期间无污泥从反应器排出。在经过0.15 g O3/g MLSS剂量的处理后，减少了约70的混合液悬浮固体（MLSS），从中释放了约1000mg/L的可溶性化学需氧量（SCOD），100mg/L的TN和18mg/L TP。表S1（补充资料）显示的MLSS，SCOD，TN和TP在污泥臭氧氧化处理前后的变化。结果显示，获得高达73.5MLSS减少率，这表明，在实验室中的培养污泥相较于从污水处理厂所取的实际污泥更容易被氧化。这些差异可能是由于使用合成废水培养实验室污泥所造成的。平均进水COD，TN和TP见表1。利用高效液相色谱法确定在进水有机酸水平。HPLC法测定的结果表明，反应器2比1的进水含有更多的有机酸，数据未能显示这些有机酸是否来自于臭氧氧化污泥。活性污泥的固体活性污泥停留时间（SRT）约15天和水力停留时间（HRT）为10小时。在生物反应池溶解氧（DO）控制在3mgO 2/L，沉淀池低于0.1mgO 2/L。沉淀池鱼生物反应池间的污泥内循环（R）体积比（回流比）为0.75（V / V）。为了要达到一个长时间稳定的状态，两个不同的反应器总运行时间大于120天不进行温度控制的运行时间。（2008年4月至8月）。在最初的50天（从5月26日开始），由原来的22-27提升到21-30，第60天后温度保持在约29（图S1）。2.2. Chemical analysis of the reactorsDuring the 122 days of operation of the two reactors, 50 mL sludge samples were withdrawn three times a week and centrifuged(12,000g, 10 min). The pellets were firstly heated at 105 to examine the MLSS concentration, and then heated at 550 to measure the inorganic suspended solids concentrations. The concentration of the mixed liquor volatile suspended solids (MLVSS) concentration was thus deduced from the two measurements. The effluent COD, TN and TP were measured three times a week. The effluent suspended solid (SS) concentrations were measured by sampling 400 mL of effluent from the outlet of the settling tanks. The COD, TN and TP concentrations were determined according to the Chinese SEPA Standard Methods (House, 2002). The relative analytical errors for these parameters were 15%. The organic acid content was determined by high-pressure liquid chromatography (HPLC-10A, Shimadzu) using an SCR-102G organic acid analysis column (Shimadzu) and an RID-10A detector (Shimadzu) at a temperature of 40 . The mobile phase was 0.1% perchloric acid aqueous solution.2.2反应器的化学分析在两个反应器运行的122天中，每周三次取50毫升的污泥样品进行离心分离（12000克，10分钟）。离心分离出的小球首先在105下研究MLSS浓度，然后加热到550到测量无机固体悬浮物浓度。从这两个测量推断混合液挥发性悬浮固体（MLVSS）的浓度。每周测三次出水COD，TN和TP。出水悬浮固体（SS）浓度测量取从沉淀池出口的 400毫升污水。采用中国国家环保总局的标准方法测定COD，TN和TP的浓度(House, 2002)。这些测量参数的相对误差为 1-5。采用高压液相色谱仪(HPLC-10A, Shimadzu)测定有机酸含量，在温度为40条件下使用SCR - 102G有机酸分析柱(Shimadzu)和RID - 10A检测器（日本岛津）。流动相为0.1高氯酸水溶液。2.3. Biochemical and microbial community analysis of the reactorsThe biological activity of the activated sludge in the two reactors was analyzed twice a week. Specifically, the protease activity, intracellular ATP concentration, total catalase (CA) activity and superoxide dismutase (SOD) activity were evaluated. The protease activity was determined using a method that has been reported elsewhere (Kim et al., 2002). One unit of the enzyme activity was defined as the amount of the enzyme that degraded 1 mg of azocasein in 60 min at 37 . The CA activity of sludge was determined using the procedure described by (Dallmier and Martin, 1988), with one unit of CA activity being defined as the amount required to decompose 1mol of H2O2 per min at 25 and pH 7. The SOD activity was measured using a SOD enzyme activity kit (Jiancheng Technology, China). One unit of SOD activity was defined as the amount required to inhibit the rate of cytochrome c reduction by 50% (Fisher et al., 2000). The intracellular ATP concentration was measured with an ATP detection kit (Biyuantian Technology, China).To evaluate the bacterial population, samples were analyzed by PCRDGGE once a week. Briefly, 2 mL of fresh sludge were collected from each bioreactor and the total DNA was then extracted using t