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我国现有近5 000座市政污水处理厂,年处理污水总量超6×1010 m3。市政污水经活性污泥法处理后,其中约1/3的有机污染物可被完全氧化成二氧化碳(CO2)。而其余大部分污水有机物则经由活性污泥代谢后被转化为微生物生物质(按污水处理量及COD折算约合污染物近1×107 t),成为市政污水处理厂产量最大的副产物——剩余污泥的主要干物质成分。剩余污泥中的有机物是生产能源、燃料和高附加值化学品的潜在原料,可被资源化利用[1-2],故不宜简单地归类于亟需处理或处置的“有害废物”。
厌氧消化(anaerobic digestion, AD)被认为是实现剩余污泥资源化的主流技术之一[2]。然而,受限于停留时间长、沼气产率低、资源化产品单一等因素,该技术的资源回收效率与资源化产品经济价值等有待进一步提升[3]。近年来,羧酸平台引起了国内外研究者的广泛关注,其“构筑模块”为短链脂肪酸(short-chain fatty acids, SCFAs, C1~C5)。SCFAs可进一步转化为多种高附加值化学品,包括酯类、生物塑料(聚羟基脂肪酸酯)、单细胞蛋白以及中链脂肪酸(medium-chain fatty acids, MCFAs)[2]。SCFAs是污泥厌氧消化的中间产物。经过水解和发酵过程,污泥中的有机物(蛋白质、多糖、脂类)被代谢转化生成SCFAs,转化率可达约70%[2, 4]。相较于传统AD产甲烷过程,污泥的厌氧发酵产SCFAs过程的停留时间、稳定性及发酵产品产率等条件均更具优势。
MCFAs是指碳原子个数为6~12的饱和脂肪酸,包括己酸(C6)、庚酸(C7)、辛酸(C8)等,在精细化工中被广泛用于制作香料、医药、化妆品及增塑剂、橡胶等化工产品。MCFAs的水溶性较弱,如己酸和辛酸在常温常压条件下的水溶解度仅为10.82 g∙L−1和0.68 g∙L−1,易于从发酵液中分离回收。碳链延长(chain elongation, CE)是微生物合成MCFAs的重要代谢途径。该过程以SCFAs为电子受体、乙醇或乳酸为电子供体进行逆向β-氧化(reverse β-oxidation,RBO)[5]。近年来,利用SCFAs产MCFAs的研究已由早期的纯培养(pure culture)体系(以人工培养基为底物接种科氏梭菌Clostridium kluyveri)向开放式培养(open culture)体系(以未经灭菌的实际有机废水或有机废弃物为底物培养混合微生物菌种)稳步推进。AGLER等[6]将发酵液pH稳定控制在5.5,利用玉米生物乙醇发酵液(富含乙醇、葡萄糖、酵母菌细胞及少量残留玉米粒生物质)产SCFAs同时进行碳链延长,并在发酵反应器下游设置在线液液萃取体系同步连续回收发酵液中的己酸,产率达到2 g∙(L∙d)−1。以此为基础,GE等[7]在550 d的连续培养过程中将己酸产率继续提高至3.4 g∙(L∙d)−1。KUCEK等[8]利用葡萄酒粗酒泥(乙醇质量分数为40%)产MCFAs,在优化条件下(pH为5.2,以COD计的有机负荷率5.8 g∙(L∙d)−1),己酸和辛酸的总产率为3.9 g∙(L∙d)−1。除了酿酒工业产生的有机废物,合成气发酵液、餐厨垃圾、乳清废水等也被报道用作产MCFAs的原料[9-13]。综上所述,目前微生物CE技术产MCFAs的主要原料均为具有高COD且易生物降解的有机废水或废弃物,而利用市政污水处理厂剩余污泥产MCFAs的系统研究仍需深入开展。
相较于酿酒废水或餐厨垃圾,污泥的惰性组分含量较高,且经由AD过程产MCFAs的产率极低。影响污泥产MCFAs的因素包括:1)污泥水解(限速步骤)进程缓慢;2)污泥厌氧发酵累积SCFAs过程中自发产甲烷导致碳转化效率降低;3)CE微生物组驯化期较为漫长。
本研究以酿酒废水厌氧塔颗粒污泥为接种物,采用混合SCFAs为电子受体、乙醇为电子供体,通过优化水力停留时间(HRT)与醇酸比进行微生物驯化以获得在代谢功能上占主导优势的CE微生物组。同时,采用碱性热水解预处理技术促进污泥破解,进而加速水解效率、提高SCFAs产率,完成从人工配制培养基溶液到真实污泥发酵液的底物转变,以“两相发酵”工艺实现污泥产MCFAs,并基于微生物多样性和宏基因组分析进一步揭示了过程中的微生态机制,以期为污泥有机质转化高附加值化学品的策略构建提供参考。
利用富乙酸剩余污泥厌氧发酵液产中链脂肪酸
Conversion of acetate-rich waste activated sludge anaerobic fermentation liquor into medium-chain fatty acids
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摘要: 为实现剩余污泥的资源化利用,探索了混菌体系中以剩余污泥为底物连续产中链脂肪酸(MCFAs)的可行性。本研究基于乙醇/乙酸人工配制废水,采用热碱水解污泥-短期厌氧发酵-微生物碳链延长(CE)反应的“两相发酵”技术合成MCFAs,并逐步优化水力停留时间(HRT)与底物醇酸比以驯化厌氧污泥微生物。结果表明:在为期135 d的连续模式CE过程中,在醇酸比为2∶1的条件下,驯化期(Phase Ⅰ~Ⅲ)的HRT由20 d逐步缩减至5 d后,典型CE微生物Clostridium sensu stricto_12成为优势菌种,其相对丰度升至65.21%,但己酸产率仅为775 mg∙(L∙d)−1;当醇酸比提高至3∶1 (Phase Ⅳ),己酸产率升至1 402 mg∙(L∙d)−1,MCFAs产物选择性明显提高。将实验期(Phase Ⅴ)系统中的底物置换为污泥厌氧发酵液,己酸产率依然稳定保持在1 400 mg∙(L∙d)−1,表明功能微生物组的结构稳定。宏基因组分析结果显示,逆向β-氧化(RBO)和脂肪酸生物合成(FAB)代谢通路均参与了CE过程的MCFAs合成;另外,相较于乙醇/乙酸人工配制废水,污泥发酵液可提高这2种代谢通路的关键酶丰度。本研究证实了污泥连续发酵产MCFAs的可行性,并阐明了过程中微生物的生态功能机制,可为污泥资源化利用提供参考。Abstract: To determine the feasibility of producing medium-chain fatty acids (MCFAs) from waste activated sludge in mixed culture, this study firstly optimized bioreactor parameters, i.e. hydraulic retention time (HRT) and ethanol : acid ratio, to assimilate anaerobes and later adopted the “two-stage fermentation” strategy, in which alkaline pretreated sludge was subjected to short-term acidogenesis and microbial chain elongation The results showed that during the 135-day chain elongation (CE) over the long term, with ethanol∶acid ratio=2∶1, when HRT was shortened from 20 d to 5 d (Phase Ⅰ~Ⅲ), the CE functional microbe, Clostridium sensu stricto_12, evolved as the dominant genus (relative abundance 65.21%). However, the maximal productivity of n-caproate was merely 775 mg∙(L∙d)−1. Subsequently, with ethanol∶acid ratio increased to 3∶1 (Phase Ⅳ), the productivity of n-caproate boosted to 1 402 mg∙(L∙d)−1, demonstrating an increased product selectivity towards MCFAs. In PhaseⅤ (test phase), during which the substrate swamped from synthetic ethanol/acetate wastewater to sludge fermentation liquor (SFL), the n-caproate productivity maintained at 1 400 mg∙(L∙d)−1. Based on the metagenomics analysis, both reverse β-oxidation (RBO) and fatty acid biosynthesis (FAB) pathways were involved in microbial chain elongation for MCFAs production. Moreover, as compared to synthetic ethanol/acetate wastewater, SFL increased the relative abundance of some key functional enzymes for the RBO and FAB pathways. The present study provided the practical evidence for continuous production of MCFAs from waste activated sludge, and more importantly, it elucidated the microbial and ecological mechanisms. Taken together, it shed a light on the sludge-derived value-added chemicals for its valorization.
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表 1 市政污水处理厂剩余污泥的基本理化性质
Table 1. Characteristics of waste activated sludge from a municipal wastewater treatment plant
pH TS/(g∙L−1) VS/(g∙L−1) COD/(mg∙L−1) SCOD/(mg∙L−1) TKN/(mg∙L−1) TAN/(mg ∙L−1) 6.72±0.05 29.66±0.60 18.63±0.38 21 100±420 346±46 2 560±94 12.4±1.5 表 2 不同运行周期的反应器运行条件
Table 2. Operational conditions of different phases of bioreactor for chain elongation
运行周期 HRT/d 醇酸比 运行时间/d Phase Ⅰ 20 2∶1 0~20 Phase Ⅱ 10 2∶1 21~50 Phase Ⅲ 5 2∶1 51~90 Phase Ⅳ 5 3∶1 91~120 Phase Ⅴ 5 3∶1 121~135 表 3 驯化期不同运行条件下的总烷基量、平均碳链长度与碳效率
Table 3. Total alkyl groups, average chain length and carbon conversion efficiency of microbial chain elongation process under different operational conditions during the acclimation period
运行周期 总烷基量/(mmol·L−1) 平均碳链长度 碳效率/% Phase Ⅰ 291.8 6.82 33.4 Phase Ⅱ 543.4 6.83 62.1 Phase Ⅲ 507 5.63 72.9 Phase Ⅳ 535.6 6.51 64.8 表 4 碳链延长(CE)过程涉及到的关键酶及其在Phase Ⅳ和Phase Ⅴ阶段微生物组中的丰度
Table 4. Key enzymes involved in chain elongation pathways and their abundance in Phase Ⅳ and Ⅴ
关键酶的名称 缩写 EC# 功能描述 不同周期酶丰度 Phase Ⅳ Phase Ⅴ Thiolase TLA 2.3.1.16 Acetyl-CoA C-acyltransferase 1 356 5 220 Ketoacycl-CoA reductase KCR 1.1.1.36 Acetyl-CoA reductase 536 4 902 Hydroxyacyl-CoA dehydratase HCD 1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase 19 452 16 412 1.1.1.35 3-hydroxyacyl-CoA dehydrogenase 5 686 15 354 2.3.1.16 Acetyl-CoA C-acyltransferase 1 356 5 220 4.2.1.17 Enoyl-CoA hydratase 13 918 15 978 Enoyl-CoA reductase ECR 5.1.2.3 3-hydroxybutyryl-CoA epimerase 666 4 922 Thioesterase TES 3.1.2.23 4-hydroxybenzoyl-CoA thioesterase 2 740 1 968 Acetyl-CoA carboxylase ACC 6.3.4.14 Biotin carboxylase 21 802 12 782 6.4.1.2 Acetyl-CoA carboxylase 40 616 33 858 Malonyltransferase MAT 2.3.1.39 [acyl-carrier-protein]S-
malonyltransferase15 826 17 026 Ketoacyl-ACP synthase KAS 2.3.1.41 β-ketoacyl-[acyl-carrier-protein] synthase Ⅰ 5 360 11 046 2.3.1.179 β-ketoacyl-[acyl-carrier-protein] synthase Ⅱ 20 000 26 892 2.3.1.180 β-ketoacyl-[acyl-carrier-protein] synthase Ⅲ 32 110 30 154 Ketoacyl-ACP reductase KAR 1.1.1.100 3-oxoacyl-[acyl-carrier-protein] reductase 32 462 40 198 Hydroxyacyl-ACP dehydratase HAD 4.2.1.59 3-hydroxyacyl-[acyl-carrier-protein] dehydratase 8 812 7 466 Enoyl-ACP reductase EAR 1.3.1.9 enoyl-[acyl-carrier-protein] reductase (NADH) 14 468 16 656 1.3.1.10 enoyl-[acyl-carrier-protein] reductase (NADPH, Si-specified) 1 448 6 510 -
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