同步厌氧脱氮除硫工艺及微生物学特性的研究
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摘要
随着工农业的发展与人类生活水平的提高,废水产生量不断增加。其中,许多工业废水、农业污水和城市污水中都有含硫污染物,废水排放所致的硫素污染严重威胁着人类健康和生态安全。这类废水的有效治理技术已成为环保界急需攻克的难题。研究证明,一些微生物能够以硝酸盐为电子受体将硫化物氧化成单质硫。据此,笔者提出了用废水生物处理系统中硝化段产生的硝酸盐来氧化厌氧段产生的硫化物,达到氮硫同时去除的设想,并对同步厌氧生物脱氮除硫工艺的运行性能和微生物特性进行了较为全面而深入的研究,主要结论如下：
1)研发了自养型同步厌氧生物脱氮除硫工艺。该工艺启动时间较短,运行26天,硫化物和硝酸盐的容积去除率分别达到文献报道水平；该工艺容积效率较高,稳态运行时,容积硫化物去除速率和容积硝酸盐去除速率分别为3.73kg·m-3·d-1和0.80 kg·m-3.d-1；该工艺基质耐受性较好,当进水硫化物浓度高于580mg·L-1时,硫化物去除率仍可保持在90%以上；该工艺水力适应性较强,当HRT从1天缩短到0.13天时,硫化物去除率保持在99%以上,硝酸盐去除率保持在92%以上。
2)优化了同步厌氧生物脱氮除硫工艺的操作条件。
①揭示了不同氮素基质对工艺性能的影响。以硝酸盐作为电子受体优于亚硝酸盐。硝酸盐型脱氮除硫工艺所能耐受的最大进水硫化物浓度和硫化物去除速率均优于亚硝酸盐型脱氮除硫工艺。同步脱氮除硫污泥对电子供体的耐受性强于电子受体。以灵敏度比作为判据,同步脱氮除硫菌受硫化物的影响不显著；而受硝酸盐／亚硝酸盐的影响较大,其中受亚硝酸盐的影响更大。同步脱氮除硫污泥对亚硝酸盐的亲和力略高于硝酸盐,其半饱和常数分别为0.26±0.08 mg·L-1和0.35±0.09 mg·L-1。
②揭示了硫氮比对工艺性能的影响。从高效性、稳定性及选择性方面看,单质硫型厌氧生物同步脱氮除硫工艺(硫氮摩尔比为5：2)明显优于混合型和硫酸盐型(硫氮摩尔比分别为5：5和5：8)。在所试范围内,单质硫型厌氧生物同步脱氮除硫工艺效能明显高于混合型和硫酸盐型,其硫化物和硝酸盐的容积去除速率高达4.86 kg·m-3·d-1和0.99 kg·m-3·d-1。单质硫型脱氮除硫工艺稳定性好,各项出水指标的波动相对较小。将硫氮摩尔比调控在较高水平(如5：2),可提高同步厌氧生物同步脱氮除硫反应对单质硫和氮气的选择性。且在所试的3种进水硫氮摩尔比下,实际反应的硫氮摩尔比有靠拢5：2的趋势。③揭示了pH和碱度对工艺运行性能的影响。控制反应液pH在7.0±0.1范围时的容积效能高于控制进水pH时的相应值。维持反应所需的中性条件时,碱度宜控制在454.1±40.5 mg CaCO3·L-1。反应过程中的碱度变化(增量)可以指示反应器内主导反应的类型及其反应进度。单质硫型、硫酸盐型和混合型生物脱氮除硫反应的硫化物去除量与碱度减少量之比分别为2.27、2.00和5.00。④揭示了冲击负荷对工艺运行性能的影响。同步厌氧生物脱氮除硫反应器对基质浓度冲击的响应过程可分为冲击期、惯性期和恢复期。其中出水硫化物浓度对基质浓度冲击的响应较为灵敏,可用作反应器性能变化的指示参数。同步厌氧生物脱氮除硫反应器对基质浓度冲击的响应与其冲击强度有关,出水pH值及基质浓度显著升高,且各性能指标的响应强度与冲击强度呈正相关。同步厌氧生物脱氮除硫反应器对基质浓度的冲击具有良好的恢复能力,所需的恢复时间短于30 h(7.5 HRT)。
3)研究了同步厌氧生物脱氮除硫工艺的微生物学特性。
①考察了同步脱氮除硫反应器稳态运行时的污泥特性与微生物生态学特性。同步脱氮除硫污泥沉降性能优良。在稳态运行的同步脱氮除硫反应器中,污泥粒径在0.54-3.99 mm之间,其中62.5%污泥直径在1.67-2.83 mm范围。颗粒污泥湿密度为1.08 kg·m-3,沉降速度在56.13-171.43 m·h-1之间。同步脱氮除硫污泥结构性能优良。同步脱氮除硫颗粒污泥由污泥亚单位(菌胶团和絮体污泥)复合而成,污泥表面微生物以杆菌为主,内部微生物形态多样。同步脱氮除硫污泥生物种群丰富。运用PCR-DGGE技术分析表明,同步脱氮除硫污泥中特征性条带较多,微生物种类多样性较高,其中以变形菌门的微生物为主。
②分离研究了脱氮除硫功能菌。从长期运行的脱氮除硫污泥中,分离获得了两个菌株(菌株CB和菌株CS),经形态学观察和16S rDNA序列比对,将其归入芽孢杆菌属,菌株CB与Bacillus pseudofirmus OF4最为接近,菌株CS与Bacillus hemicellulosilytus、Bacillus halodurans最为接近。首次试验证明芽孢杆菌菌株具有脱氮除硫功能,其中菌株CB对硝酸盐、硫化物的转化能力及亲和力大于菌株CS。经Biolog板检测,菌株CB的基质多样性不明显,菌株CS则可利用Biolog板中多种碳源。
With the development of economy and the improvement of standard living, large amount of wastewaters are generated. The wastewaters from industry, agriculture and housing settlements contain sulfur compounds. Sulfur pollution has already posed hazardous effects on human health and ecological safety. Hence, how to treat such wastewater economically and efficiently is one of the most popular environmental topics in recent years. It has been shown that some bacterial species like Thiobacillus denitrificans can oxidize sulfide to elemental sulfur simultaneously reducing nitrate to dinitrogen. For such reasons, the simultaneous anaerobic sulfide and nitrate removal process has been developed. In this research, the process was studied from operating conditions and microbial properties. Major research results are as follows:
1) The simultaneous anaerobic sulfide and nitrate removal process in inorganic condition was studied. It was discovered that the start-up course of the process was fast by using Upflow Anaerobic Sludge Blanket (UASB) reactor. The volumetric removal rates of sulfide and nitrate were 0.323 kg·m-3·d-1 and 0.071 kg·m-3·d-1 after 26 days'operation, which had reached the reported level in liteature. The results also showed that the process could hold a high sulfide and nitrate removal loading rate of 3.73 kg·m-3·d-1 and 0.80 kg·m-3·d-1, respectively, under steady state. It was capable of tolerating high influent substrate concentration (580 mg-L-1 and 110 mg-L-1). It was capable of tolerating short hydraulic retention time (HRT). When HRT was 0.13 d, the removal percentage of sulfide and nitrate were higher than 99% and 92%, respectively.
2) The operating conditions of simultaneous anaerobic sulfide and nitrate removal process were investigated.
①The effect of influent substrates on the performance of simultaneous anaerobic sulfur and nitrogen removal process was investigated. It was discovered that the process using nitrate as electron accepter was better than that using nitrite as electron accepter. The maximum acceptable sulfide concentration and volumetric removal rate of simultaneous anaerobic sulfide and nitrate removal process were higher than those of simultaneous anaerobic sulfide and nitrite removal process. The sludge had better tolerance to electron donor than electron accepter. Judged by sensitivity ratio, the activated sludge was more tolerant to sulfide, while no significant differences were found in respect of using nitrate or nitrite as influent substrate. The activated sludge was less tolerant to nitrate, the least tolerant to nitrite. The sludge had slightly better affinity on nitrite than nitrate, whose half saturation values were 0.26±0.08 mg·L-1 and 0.35±0.09 mg·L-1, respectively. The minimum reaction time (5h) for nitrate and sulfide was relatively longer than that for nitrite and sulfide as influent substrates (4 h).
②The effect of sulfide to nitrate (S/N) molar ratio on the performance of simultaneous anaerobic sulfide and nitrate removal process was investigated. It was discovered that the process performance at molar S/N ratio of 5:2 was significantly better than those at S/N molar ratios of 5:5 and 5:8, based on capacity, stability and selectivity. The volumetric sulfide and nitrate removal rates at molar S/N ratio of 5:2 were 4.86 kg·m-3·d-1 and 0.99 kg·m-3·d-1, respectively, which were higher than those at the other S/N molar ratios. Moreover, the fluctuations in the effluent at S/N ratio of 5:2 were less than those at the other two tested ratios, which showed better stability. The selectivity for elemental sulfur and dinitrogen was improved when the S/N molar ratio was set at 5:2 rather than 5:5 or 5:8. Moreover, the ratio of converted sulfide to converted nitrate tended to approach 5:2 during the operation.
③The effect of pH and alkalinity on the performance of simultaneous anaerobic sulfide and nitrate removal process was investigated. It was discovered that the process performance was better when the reaction pH was controlled between 6.9 and 7.1 than that when the influent pH was controlled between 7.5 and 8.0. In order to keep a neutral condition, the alkalinity should be adjusted at 454.1±40.5 mg CaCO3·L-1. The alkalinity change in the process can indicate the style and extent of the dominant reaction. When S/N ratios were 5:2,5:8 and 5:5, the ratios of removed sulfide to decreased alkalinity were 2.98,2.56 and 6.41, respectively.
④The effect of substrate shock on the performance of simultaneous anaerobic sulfide and nitrate removal process was investigated. It was discovered that based on the response to the shock loads, the reactor performance could be divided into three stages:disturbance, inertia and recovery periods. The effluent sulfide concentration was a sensitive parameter, which increased up to 18 times of that at steady state and could be used as an indicator of the reactor performance. The effect of the shock loads on the reactor performance was related to the intensity of the shock loads. The responsive strength increased with the increasing intensity of shock loads. The performance was able to recover from disturbances at all the tested shock loads. In 30 hours (7.5h), all the performance indices of the reactor could return to the initial stable state.
3) The microbial properties of simultaneous anaerobic sulfide and nitrate removal process were studied.
①The physical and ecological characteristics of cultivated sludge for simultaneous anaerobic sulfide and nitrate removal were studied. It was discovered that the cultivated sludge showed good settlability, whose diameters, settling velocities and density were 0.54-3.99 mm,56.13-171.43 m·h-1 and 1.08 kg·m-3, respectively. It was irregular-shaped and was composed of subunits (such as zoogloea and floc) under optical microscope. It was observed that the dominant microorganisms on the surface were bacilli, but those in the inner were diversiform under scanning electron microscope, The results from PCR-DGGE analysis exhibited rich diversity of microbial populations and dominant kinds of microorganisms Proteobacteria.
②The bacteria responsible for simultaneous sulfide and nitrate removal were isolated and identified. It was discovered that the bacterial strains CB and CS were isolated from the UASB reactor working under steady-state with sulfide and nitrate as electron donor and acceptor, respectively. Based on electron microscopy, physiological test and 16S rDNA phylogenetic sequence analysis, the isolate CB was very close to Bacillus pseudofirmus and the isolate CS was very close to Bacillus hemicellulosilytus and Bacillus halodurans. Both the isolate CB and the isolate CS are able to use nitrate for sulfide oxidation. The isolate CS has greater capability to oxidize sulfide with nitrate as electron acceptor. According to Biolog carbon source utilization test, the isolate CB can only use a few of carbon sources, while the isolate CB can use a lot of carbon sources. Some carbon sources, such as glucose, galactose, arabinose, can be used to promote the growth of these autotrophic bacteria.
1)研发了自养型同步厌氧生物脱氮除硫工艺。该工艺启动时间较短,运行26天,硫化物和硝酸盐的容积去除率分别达到文献报道水平；该工艺容积效率较高,稳态运行时,容积硫化物去除速率和容积硝酸盐去除速率分别为3.73kg·m-3·d-1和0.80 kg·m-3.d-1；该工艺基质耐受性较好,当进水硫化物浓度高于580mg·L-1时,硫化物去除率仍可保持在90%以上；该工艺水力适应性较强,当HRT从1天缩短到0.13天时,硫化物去除率保持在99%以上,硝酸盐去除率保持在92%以上。
2)优化了同步厌氧生物脱氮除硫工艺的操作条件。
①揭示了不同氮素基质对工艺性能的影响。以硝酸盐作为电子受体优于亚硝酸盐。硝酸盐型脱氮除硫工艺所能耐受的最大进水硫化物浓度和硫化物去除速率均优于亚硝酸盐型脱氮除硫工艺。同步脱氮除硫污泥对电子供体的耐受性强于电子受体。以灵敏度比作为判据,同步脱氮除硫菌受硫化物的影响不显著；而受硝酸盐／亚硝酸盐的影响较大,其中受亚硝酸盐的影响更大。同步脱氮除硫污泥对亚硝酸盐的亲和力略高于硝酸盐,其半饱和常数分别为0.26±0.08 mg·L-1和0.35±0.09 mg·L-1。
②揭示了硫氮比对工艺性能的影响。从高效性、稳定性及选择性方面看,单质硫型厌氧生物同步脱氮除硫工艺(硫氮摩尔比为5：2)明显优于混合型和硫酸盐型(硫氮摩尔比分别为5：5和5：8)。在所试范围内,单质硫型厌氧生物同步脱氮除硫工艺效能明显高于混合型和硫酸盐型,其硫化物和硝酸盐的容积去除速率高达4.86 kg·m-3·d-1和0.99 kg·m-3·d-1。单质硫型脱氮除硫工艺稳定性好,各项出水指标的波动相对较小。将硫氮摩尔比调控在较高水平(如5：2),可提高同步厌氧生物同步脱氮除硫反应对单质硫和氮气的选择性。且在所试的3种进水硫氮摩尔比下,实际反应的硫氮摩尔比有靠拢5：2的趋势。③揭示了pH和碱度对工艺运行性能的影响。控制反应液pH在7.0±0.1范围时的容积效能高于控制进水pH时的相应值。维持反应所需的中性条件时,碱度宜控制在454.1±40.5 mg CaCO3·L-1。反应过程中的碱度变化(增量)可以指示反应器内主导反应的类型及其反应进度。单质硫型、硫酸盐型和混合型生物脱氮除硫反应的硫化物去除量与碱度减少量之比分别为2.27、2.00和5.00。④揭示了冲击负荷对工艺运行性能的影响。同步厌氧生物脱氮除硫反应器对基质浓度冲击的响应过程可分为冲击期、惯性期和恢复期。其中出水硫化物浓度对基质浓度冲击的响应较为灵敏,可用作反应器性能变化的指示参数。同步厌氧生物脱氮除硫反应器对基质浓度冲击的响应与其冲击强度有关,出水pH值及基质浓度显著升高,且各性能指标的响应强度与冲击强度呈正相关。同步厌氧生物脱氮除硫反应器对基质浓度的冲击具有良好的恢复能力,所需的恢复时间短于30 h(7.5 HRT)。
3)研究了同步厌氧生物脱氮除硫工艺的微生物学特性。
①考察了同步脱氮除硫反应器稳态运行时的污泥特性与微生物生态学特性。同步脱氮除硫污泥沉降性能优良。在稳态运行的同步脱氮除硫反应器中,污泥粒径在0.54-3.99 mm之间,其中62.5%污泥直径在1.67-2.83 mm范围。颗粒污泥湿密度为1.08 kg·m-3,沉降速度在56.13-171.43 m·h-1之间。同步脱氮除硫污泥结构性能优良。同步脱氮除硫颗粒污泥由污泥亚单位(菌胶团和絮体污泥)复合而成,污泥表面微生物以杆菌为主,内部微生物形态多样。同步脱氮除硫污泥生物种群丰富。运用PCR-DGGE技术分析表明,同步脱氮除硫污泥中特征性条带较多,微生物种类多样性较高,其中以变形菌门的微生物为主。
②分离研究了脱氮除硫功能菌。从长期运行的脱氮除硫污泥中,分离获得了两个菌株(菌株CB和菌株CS),经形态学观察和16S rDNA序列比对,将其归入芽孢杆菌属,菌株CB与Bacillus pseudofirmus OF4最为接近,菌株CS与Bacillus hemicellulosilytus、Bacillus halodurans最为接近。首次试验证明芽孢杆菌菌株具有脱氮除硫功能,其中菌株CB对硝酸盐、硫化物的转化能力及亲和力大于菌株CS。经Biolog板检测,菌株CB的基质多样性不明显,菌株CS则可利用Biolog板中多种碳源。
With the development of economy and the improvement of standard living, large amount of wastewaters are generated. The wastewaters from industry, agriculture and housing settlements contain sulfur compounds. Sulfur pollution has already posed hazardous effects on human health and ecological safety. Hence, how to treat such wastewater economically and efficiently is one of the most popular environmental topics in recent years. It has been shown that some bacterial species like Thiobacillus denitrificans can oxidize sulfide to elemental sulfur simultaneously reducing nitrate to dinitrogen. For such reasons, the simultaneous anaerobic sulfide and nitrate removal process has been developed. In this research, the process was studied from operating conditions and microbial properties. Major research results are as follows:
1) The simultaneous anaerobic sulfide and nitrate removal process in inorganic condition was studied. It was discovered that the start-up course of the process was fast by using Upflow Anaerobic Sludge Blanket (UASB) reactor. The volumetric removal rates of sulfide and nitrate were 0.323 kg·m-3·d-1 and 0.071 kg·m-3·d-1 after 26 days'operation, which had reached the reported level in liteature. The results also showed that the process could hold a high sulfide and nitrate removal loading rate of 3.73 kg·m-3·d-1 and 0.80 kg·m-3·d-1, respectively, under steady state. It was capable of tolerating high influent substrate concentration (580 mg-L-1 and 110 mg-L-1). It was capable of tolerating short hydraulic retention time (HRT). When HRT was 0.13 d, the removal percentage of sulfide and nitrate were higher than 99% and 92%, respectively.
2) The operating conditions of simultaneous anaerobic sulfide and nitrate removal process were investigated.
①The effect of influent substrates on the performance of simultaneous anaerobic sulfur and nitrogen removal process was investigated. It was discovered that the process using nitrate as electron accepter was better than that using nitrite as electron accepter. The maximum acceptable sulfide concentration and volumetric removal rate of simultaneous anaerobic sulfide and nitrate removal process were higher than those of simultaneous anaerobic sulfide and nitrite removal process. The sludge had better tolerance to electron donor than electron accepter. Judged by sensitivity ratio, the activated sludge was more tolerant to sulfide, while no significant differences were found in respect of using nitrate or nitrite as influent substrate. The activated sludge was less tolerant to nitrate, the least tolerant to nitrite. The sludge had slightly better affinity on nitrite than nitrate, whose half saturation values were 0.26±0.08 mg·L-1 and 0.35±0.09 mg·L-1, respectively. The minimum reaction time (5h) for nitrate and sulfide was relatively longer than that for nitrite and sulfide as influent substrates (4 h).
②The effect of sulfide to nitrate (S/N) molar ratio on the performance of simultaneous anaerobic sulfide and nitrate removal process was investigated. It was discovered that the process performance at molar S/N ratio of 5:2 was significantly better than those at S/N molar ratios of 5:5 and 5:8, based on capacity, stability and selectivity. The volumetric sulfide and nitrate removal rates at molar S/N ratio of 5:2 were 4.86 kg·m-3·d-1 and 0.99 kg·m-3·d-1, respectively, which were higher than those at the other S/N molar ratios. Moreover, the fluctuations in the effluent at S/N ratio of 5:2 were less than those at the other two tested ratios, which showed better stability. The selectivity for elemental sulfur and dinitrogen was improved when the S/N molar ratio was set at 5:2 rather than 5:5 or 5:8. Moreover, the ratio of converted sulfide to converted nitrate tended to approach 5:2 during the operation.
③The effect of pH and alkalinity on the performance of simultaneous anaerobic sulfide and nitrate removal process was investigated. It was discovered that the process performance was better when the reaction pH was controlled between 6.9 and 7.1 than that when the influent pH was controlled between 7.5 and 8.0. In order to keep a neutral condition, the alkalinity should be adjusted at 454.1±40.5 mg CaCO3·L-1. The alkalinity change in the process can indicate the style and extent of the dominant reaction. When S/N ratios were 5:2,5:8 and 5:5, the ratios of removed sulfide to decreased alkalinity were 2.98,2.56 and 6.41, respectively.
④The effect of substrate shock on the performance of simultaneous anaerobic sulfide and nitrate removal process was investigated. It was discovered that based on the response to the shock loads, the reactor performance could be divided into three stages:disturbance, inertia and recovery periods. The effluent sulfide concentration was a sensitive parameter, which increased up to 18 times of that at steady state and could be used as an indicator of the reactor performance. The effect of the shock loads on the reactor performance was related to the intensity of the shock loads. The responsive strength increased with the increasing intensity of shock loads. The performance was able to recover from disturbances at all the tested shock loads. In 30 hours (7.5h), all the performance indices of the reactor could return to the initial stable state.
3) The microbial properties of simultaneous anaerobic sulfide and nitrate removal process were studied.
①The physical and ecological characteristics of cultivated sludge for simultaneous anaerobic sulfide and nitrate removal were studied. It was discovered that the cultivated sludge showed good settlability, whose diameters, settling velocities and density were 0.54-3.99 mm,56.13-171.43 m·h-1 and 1.08 kg·m-3, respectively. It was irregular-shaped and was composed of subunits (such as zoogloea and floc) under optical microscope. It was observed that the dominant microorganisms on the surface were bacilli, but those in the inner were diversiform under scanning electron microscope, The results from PCR-DGGE analysis exhibited rich diversity of microbial populations and dominant kinds of microorganisms Proteobacteria.
②The bacteria responsible for simultaneous sulfide and nitrate removal were isolated and identified. It was discovered that the bacterial strains CB and CS were isolated from the UASB reactor working under steady-state with sulfide and nitrate as electron donor and acceptor, respectively. Based on electron microscopy, physiological test and 16S rDNA phylogenetic sequence analysis, the isolate CB was very close to Bacillus pseudofirmus and the isolate CS was very close to Bacillus hemicellulosilytus and Bacillus halodurans. Both the isolate CB and the isolate CS are able to use nitrate for sulfide oxidation. The isolate CS has greater capability to oxidize sulfide with nitrate as electron acceptor. According to Biolog carbon source utilization test, the isolate CB can only use a few of carbon sources, while the isolate CB can use a lot of carbon sources. Some carbon sources, such as glucose, galactose, arabinose, can be used to promote the growth of these autotrophic bacteria.
引文
Alcantara S, Velasco A, Munoz A, et al. Hydrogen sulfide oxidation by amicrobial consortium in a recirculation reactor system:sulfur formation under oxygen limitation and removal of phenols[J]. Environmental Science and Technology, 2004,38:918-923.
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