不同氮源基质厌氧消化过程中氮转化及微生态的差异揭示污泥蛋白类物质的降解瓶颈

doi:10.16085/j.issn.1000-6613.2024-0745

摘要/Abstract

摘要：

针对污泥中蛋白类物质厌氧转化率低的问题，研究对比了污泥与不同氮源基质（秸秆、鸡粪、大豆、牛肉）中蛋白类物质降解性能以及系统中微生态结构的差异。结果表明，易接触蛋白类物质（pr-A）在各组系统中的厌氧降解性能均优于难接触蛋白类物质（pr-B），其中，不含有pr-B的大豆和牛肉呈现出最佳的厌氧消化性能，非游离氨基酸的水解率、挥发性脂肪酸（VFAs）积累量和甲烷产量分别是污泥的1.44~1.56倍、3.34~3.35倍、1.61~1.76倍。五组厌氧消化系统中的微生物种群结构也呈现出显著差异，其中厚壁菌门（Firmicutes）、广古菌门（Euryarchaeota）在大豆和牛肉组厌氧系统中具有显著的丰度优势，并对部分氨基酸代谢模块（M00121、M00036、M00307）的贡献度更高。蛋白类物质与厌氧系统中优势菌属的相关性分析表明，pr-A比例的增加能驱动擅长分泌水解酶和产甲烷的菌属增殖（如Syntrophomonas、Methanobacterium、Clostridium等），而高含量pr-B作为底物存在时不利于大多数菌群的增殖代谢，只能促进部分耐受性强的菌属适应较差的底物环境，同时也会间接影响pr-A的降解。因此pr-B是污泥中蛋白类物质厌氧降解的瓶颈所在，未来的强化降解研究应考虑将pr-B转化为pr-A，以增加底物的生物可及性，并有助于构建丰富活跃的微生态系统。

关键词: 污泥, 降解, 蛋白质, 氨基酸, 群落结构, 代谢

Abstract:

In response to the problem of low anaerobic conversion rate of protein substances in sludge, this study compared the degradation performance of protein substances in sludge and different nitrogen source substrates (straw, chicken manure, soybeans, beef) and the differences in the microecological structure of the systems. The results showed that the anaerobic degradation performance of easily accessible proteins (pr-A) was superior to that of difficult-to-access proteins (pr-B) in all systems. Among these, soybeans and beef, which did not contain pr-B, exhibited the best anaerobic digestion performance. The hydrolysis rates of non-free amino acids, the accumulation of volatile fatty acids (VFAs), and methane production in these systems were 1.44—1.56 times, 3.34—3.35 times, and 1.61—1.76 times higher, respectively, compared to those in the sludge system. The microbial community structures in the five anaerobic digestion systems also showed significant differences. Specifically, Firmicutes and Euryarchaeota had a notable abundance advantage in the soybean and beef anaerobic systems and contributed more significantly to certain amino acid metabolism modules (M00121, M00036, M00307). Correlation analysis between protein substances and dominant microbial genera in the anaerobic systems indicated that an increase in the proportion of pr-A could drive the proliferation of genera proficient in secreting hydrolytic enzymes and producing methane (e.g., Syntrophomonas, Methanobacterium, Clostridium, etc). In contrast, high levels of pr-B as a substrate were detrimental to the proliferation and metabolism of most microbial communities, promoting only the adaptation of a few highly tolerant genera to the suboptimal substrate environment and indirectly affecting the degradation of pr-A. Thus, pr-B represented a bottleneck in the anaerobic degradation of protein substances in sludge. Future research on enhanced degradation should focus on converting pr-B to pr-A to increase substrate bioavailability and support the development of a rich and active micro-ecosystem.

Key words: sludge, degradation, protein, amino acids, community structure, metabolism

中图分类号:

X705

高君, 孙晓杰, 董滨. 不同氮源基质厌氧消化过程中氮转化及微生态的差异揭示污泥蛋白类物质的降解瓶颈[J]. 化工进展, 2025, 44(7): 4190-4201.

GAO Jun, SUN Xiaojie, DONG Bin. Differences in nitrogen conversion and microecology during anaerobic digestion of different nitrogen-source substrates reveal the degradation bottleneck of sludge protein substances[J]. Chemical Industry and Engineering Progress, 2025, 44(7): 4190-4201.

图/表 11

表1 厌氧消化接种泥和基质的性质

接种泥和基质	含水率/%	(VS/TS)/%	pH
接种泥	93.26±0.01	43.99±0.01	7.89±0.05
污泥	88.4±0.03	55.78±0.02	7.74±0.03
秸秆	9.36±0.02	73.07±0.01	7.58±0.02
鸡粪	85.77±0.01	61.68±0.05	7.76±0.03
大豆	87.01±0.03	94.40±0.01	7.79±0.02
牛肉	89.81±0.05	95.96±0.07	7.81±0.02

图1 不同氮源基质的累积厌氧产甲烷量和厌氧消化前后蛋白类物质的分布情况

图2 污泥、秸秆、鸡粪、大豆、牛肉在产酸发酵过程中挥发性脂肪酸（VFAs）含量和分布

表2 五种厌氧消化系统中氨基酸的分布变化情况

项目	氨基酸含量/μg·mL^-1
项目	污泥	秸秆	鸡粪	大豆	牛肉
厌氧消化前
水解氨基酸	3694.56	1084.96	3873.02	4398.65	6074.24
游离氨基酸	159.86	46.97	151.62	208.22	356.67
非游离氨基酸	3534.70	1037.98	3721.40	4190.43	5717.57
厌氧消化后
水解氨基酸	2707.76	776.07	2640.00	2109.17	2995.63
游离氨基酸	453.61	122.16	379.04	282.16	269.15
非游离氨基酸	2254.15	653.91	2260.96	1827.01	2726.48

图3 五组系统厌氧消化中期和后期的门水平微生物菌群分布和主成分分析

图4 污泥与其他基质厌氧消化系统中门水平微生物菌群的卡方检验

图5 五组厌氧消化系统中与NH3利用和产生直接相关的酶的基因丰度

表3 与NH3的利用和产生直接相关的酶及相关反应

酶编号	定义	反应
6.3.1.2	谷氨酸-氨连接酶	ATP+L-谷氨酸+NH₃ $̿$ ADP+磷酸+L-谷氨酰胺
1.7.99.1	羟胺还原酶	NH₃+H₂O+受体 $̿$ 羟胺+还原受体
1.7.2.2	亚硝酸还原酶（细胞色素；生成氨）	NH₃+2H₂O+6氧化细胞色素c $̿$ 亚硝酸盐+6还原细胞色素c+7H⁺
6.3.1.1	天冬氨酸-氨连接酶	ATP+L-天冬氨酸+NH₃ $̿$ AMP+焦磷酸+L-天冬酰胺
4.3.1.3	组氨酸氨裂解酶	L-组氨酸 $̿$ 尿氨酸+NH₃
6.3.4.2	CTP 合成酶（谷氨酰胺水解）	L-谷氨酰胺+H₂O $̿$ L-谷氨酸+NH₃
4.3.1.4	亚氨甲基四氢叶酸环脱氨酶	5-亚氨甲基四氢叶酸 $̿$ 5,10-亚甲基四氢叶酸+NH₃
2.1.2.10	氨甲基转移酶	[蛋白]-S8-氨甲基二氢硫辛酰赖氨酸+四氢叶酸 $̿$ [蛋白]-二氢硫辛酰赖氨酸+5,10-亚甲基四氢叶酸+NH₃
4.3.1.19	苏氨酸氨裂解酶	L-苏氨酸 $̿$ 2-氧代丁酸+NH₃
2.5.1.61	羟甲基胆色素合成酶	4-胆色素原+H₂O $̿$ 羟甲基胆色素+4NH₃
4.3.1.17	L-丝氨酸氨裂解酶	L-丝氨酸 $̿$ 丙酮酸+NH₃
4.3.1.1	天冬氨酸氨裂解酶	L-天冬氨酸 $̿$ 延胡索酸+NH₃
4.3.1.7	乙醇胺氨裂解酶	乙醇胺 $̿$ 乙醛+NH₃
4.3.1.2	甲基天冬氨酸氨裂解酶	L-苏氨酸-3-甲基天冬氨酸 $̿$ 美沙康酸+NH₃
4.3.1.15	二氨基丙酸氨裂解酶	2,3-二氨基丙酸+H₂O $̿$ 丙酮酸+2NH₃
4.3.1.12	鸟氨酸环脱氨酶	L-鸟氨酸 $̿$ L-脯氨酸+NH₃
4.3.1.18	D-丝氨酸氨裂解酶	D-丝氨酸 $̿$ 丙酮酸+NH₃

表3 与NH3的利用和产生直接相关的酶及相关反应

酶编号	定义	反应
6.3.1.2	谷氨酸-氨连接酶	ATP+L-谷氨酸+NH₃ $̿$ ADP+磷酸+L-谷氨酰胺
1.7.99.1	羟胺还原酶	NH₃+H₂O+受体 $̿$ 羟胺+还原受体
1.7.2.2	亚硝酸还原酶（细胞色素；生成氨）	NH₃+2H₂O+6氧化细胞色素c $̿$ 亚硝酸盐+6还原细胞色素c+7H⁺
6.3.1.1	天冬氨酸-氨连接酶	ATP+L-天冬氨酸+NH₃ $̿$ AMP+焦磷酸+L-天冬酰胺
4.3.1.3	组氨酸氨裂解酶	L-组氨酸 $̿$ 尿氨酸+NH₃
6.3.4.2	CTP 合成酶（谷氨酰胺水解）	L-谷氨酰胺+H₂O $̿$ L-谷氨酸+NH₃
4.3.1.4	亚氨甲基四氢叶酸环脱氨酶	5-亚氨甲基四氢叶酸 $̿$ 5,10-亚甲基四氢叶酸+NH₃
2.1.2.10	氨甲基转移酶	[蛋白]-S8-氨甲基二氢硫辛酰赖氨酸+四氢叶酸 $̿$ [蛋白]-二氢硫辛酰赖氨酸+5,10-亚甲基四氢叶酸+NH₃
4.3.1.19	苏氨酸氨裂解酶	L-苏氨酸 $̿$ 2-氧代丁酸+NH₃
2.5.1.61	羟甲基胆色素合成酶	4-胆色素原+H₂O $̿$ 羟甲基胆色素+4NH₃
4.3.1.17	L-丝氨酸氨裂解酶	L-丝氨酸 $̿$ 丙酮酸+NH₃
4.3.1.1	天冬氨酸氨裂解酶	L-天冬氨酸 $̿$ 延胡索酸+NH₃
4.3.1.7	乙醇胺氨裂解酶	乙醇胺 $̿$ 乙醛+NH₃
4.3.1.2	甲基天冬氨酸氨裂解酶	L-苏氨酸-3-甲基天冬氨酸 $̿$ 美沙康酸+NH₃
4.3.1.15	二氨基丙酸氨裂解酶	2,3-二氨基丙酸+H₂O $̿$ 丙酮酸+2NH₃
4.3.1.12	鸟氨酸环脱氨酶	L-鸟氨酸 $̿$ L-脯氨酸+NH₃
4.3.1.18	D-丝氨酸氨裂解酶	D-丝氨酸 $̿$ 丙酮酸+NH₃

表4 与氨基酸代谢相关基因的定义和相对丰度（%）

编号	污泥	秸秆	鸡粪	大豆	牛肉	定义
ko00250	0.8950	0.9371	0.9224	0.9328	0.9019	丙氨酸、天冬氨酸和谷氨酸代谢
ko00260	1.0005	0.9925	1.0006	1.0185	1.0249	甘氨酸、丝氨酸和苏氨酸代谢
ko00270	0.9535	0.9637	0.9559	0.9703	0.9751	半胱氨酸和蛋氨酸代谢
ko00280	0.4763	0.4768	0.4924	0.5619	0.5022	缬氨酸、亮氨酸和异亮氨酸降解
ko00281	0.0518	0.0540	0.0588	0.0818	0.0590	香叶醇降解
ko00290	0.3337	0.3755	0.3669	0.3594	0.3388	缬氨酸、亮氨酸和异亮氨酸生物合成
ko00300	0.5573	0.5391	0.5294	0.5309	0.5466	赖氨酸生物合成
ko00310	0.3434	0.3181	0.3400	0.3584	0.3486	赖氨酸降解
ko00330	0.4417	0.4805	0.4911	0.5156	0.4941	精氨酸和脯氨酸代谢
ko00340	0.3840	0.3525	0.3527	0.3446	0.3569	组氨酸代谢
ko00350	0.1891	0.1876	0.1935	0.2104	0.1978	酪氨酸代谢
ko00360	0.3048	0.3093	0.3104	0.3366	0.3104	苯丙氨酸代谢
ko00380	0.3171	0.3051	0.3239	0.3564	0.3320	色氨酸代谢
ko00400	0.5209	0.5086	0.5031	0.4952	0.4812	苯丙氨酸、酪氨酸和色氨酸生物合成
ko00410	0.1857	0.1648	0.1723	0.1855	0.1792	β-丙氨酸代谢
ko00430	0.1219	0.1339	0.1402	0.1416	0.1386	牛磺酸和假牛磺酸代谢
ko00440	0.0289	0.0389	0.0313	0.0397	0.0321	磷酸盐和磷酸亚盐代谢
ko00450	0.3992	0.3826	0.3906	0.3687	0.3890	硒化合物代谢
ko00460	0.1818	0.2019	0.1992	0.1683	0.1943	氰氨基酸代谢
ko00471	0.1728	0.1616	0.1653	0.1623	0.1706	D-谷氨酰胺和 D-谷氨酸代谢
ko00472	0.0261	0.0243	0.0265	0.0245	0.0293	D-精氨酸和 D-鸟氨酸代谢
ko00473	0.1387	0.1670	0.1601	0.1834	0.1691	D-丙氨酸代谢
ko00480	0.2111	0.2414	0.2447	0.2726	0.2498	谷胱甘肽代谢
总和	8.2350	8.3167	8.3712	8.6194	8.4214	—

图6 相对丰度排名前15的菌门对M00121、M00036和M00307模块的物种贡献度分布热图

图7 基质性质与优势菌属物种相对丰度的Spearman等级相关系数矩阵

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