纤维素降解菌的筛选以及对水稻秸秆的代谢利用

doi:10.11686/cyxb2024385

摘要/Abstract

摘要：

为提高秸秆的利用率，从白蚁肠道中通过刚果红初筛、滤纸降解复筛试验筛选到了两株纤维素降解菌，经NCBI同源序列比对百分比分别鉴定为沙福芽孢杆菌（BS）和伊朗纤维单胞菌（CE），其内切葡聚糖酶、外切葡聚糖酶、β-葡萄糖苷酶活力测定结果分别为0.102、0.321、0.112 U·mL^-1和0.202、0.434、0.131 U·mL^-1。两株菌的滤纸发酵差异代谢物主要是氨基酸及其代谢产物、苯及其衍生物、醛酮酯类、生物碱、有机酸及其衍生物和杂环化合物等物质。其差异代谢途径以苯丙氨酸、酪氨酸和色氨酸的生物合成和芳香族化合物的降解等为主。以水稻秸秆为唯一碳源培养沙福芽孢杆菌和伊朗纤维单胞菌14 d后，秸秆出现不同程度降解，与对照组相比，沙福芽孢杆菌和伊朗纤维单胞菌处理下秸秆的酸性洗涤纤维（48.80%、35.43% DM）和半纤维素含量（19.90%、17.53% DM）以及可溶性碳水化合物含量（0.11%、0.18% DM）均显著降低，伊朗纤维单胞菌处理下秸秆的失重率（43.12% DM）显著增加、中性洗涤纤维含量（52.95% DM）显著降低。

关键词: 纤维素降解菌, 滤纸, 水稻秸秆

Abstract:

To enhance the utilization rate of straw， two cellulose-degrading bacteria were isolated from the intestinal tract of termites. The bacteria were initially screened using Congo red and subsequently re-screened with a filter paper degradation test. They were identified as Cellulomonas iranensis （CE） and Bacillus safensis （BS） through NCBI homologous sequence alignment. The enzymatic activities of endoglucanase， exoglucanase， and β-glucosidase were quantified to two bacterial species. The measured activities for BS were 0.102， 0.321， and 0.112 U·mL^-1， respectively， whereas those for CE were 0.202， 0.434， and 0.131 U·mL^-1， respectively. The differential metabolites identified during filter paper fermentation by BS and CE were primarily amino acids and their metabolites， benzene and its derivatives， aldehydes， ketones， esters， alkaloids， organic acids and their derivatives， as well as heterocyclic compounds. The differential metabolic pathways were dominated by the biosynthesis of phenylalanine， tyrosine， and tryptophan， as well as the degradation of aromatic compounds. After 14 days fermentation， BS and CE were cultivated with rice （Oryza sativa） straw as the sole carbon source. The straw was degraded to varying degrees. Both of BS and CE significantly （P<0.05） reduced the contents of acid detergent fiber （48.80%， 35.43% DM）， hemicellulose （19.90%， 17.53% DM） and water soluble carbohydrate （0.11%， 0.18% DM）. In addition， the inoculation of CE also obviously increased （P<0.05） the weight loss rate of straw （43.12% DM） and reduced the content of neutral detergent fiber （52.95% DM）.

Key words: cellulose-degrading bacteria, filter paper, rice straw

陈丹丹, 王垚, 郭田心, 梁秋雨, 张庆, 骈瑞琪. 纤维素降解菌的筛选以及对水稻秸秆的代谢利用[J]. 草业学报, 2025, 34(9): 185-193.

Dan-dan CHEN, Yao WANG, Tian-xin GUO, Qiu-yu LIANG, Qing ZHANG, Rui-qi PIAN. Screening of cellulose-degrading bacteria involved in metabolic utilization of rice straw[J]. Acta Prataculturae Sinica, 2025, 34(9): 185-193.

图/表 10

图1 纤维素降解菌的刚果红染色结果a： 1号菌No.1 bacterium； b： 2号菌No.2 bacterium. 黄色圈：菌落直径（d）；红色圈：透明圈直径（D）。Yellow circle： Colony diameter （d）； Red circle： Transparent circle diameter （D）.

Fig.1 Congo red staining results of cellulose-degrading bacteria

图2 培养2 d后纤维素降解菌的滤纸降解情况a：对照组Control group； b： 1号菌No.1 bacterium； c： 2号菌No.2 bacterium.

Fig.2 Filter paper degradation of cellulose-degrading bacteria after 2 days of cultivation

图3 沙福芽孢杆菌和伊朗纤维单胞菌的革兰氏染色结果a：沙福芽孢杆菌B. safensis； b：伊朗纤维单胞菌C. iranensis.

Fig.3 Gram staining results of B. safensis and C. iranensis

表1 沙福芽孢杆菌和伊朗纤维单胞菌的生理生化结果

Table 1 Physiological and biochemical results of B. safensis and C. iranensis

项目 Item	沙福芽孢杆菌 B. safensis	伊朗纤维单胞菌 C. iranensis	项目 Item	沙福芽孢杆菌 B. safensis	伊朗纤维单胞菌 C. iranensis
革兰氏Gram	+	+	D-水杨苷D-salicylic acid glycoside	+	-
过氧化氢酶Catalase	+	+	纤维二糖Cellobiose	+	+
运动性Motile	+	+	柠檬酸盐Citrate	+	-
淀粉Starch	-	-	麦芽糖Maltose	+	+
甲基红试验Methylred test	+	+	山梨醇Sorbitol	+	+
明胶化Gelatinization	-	-	海藻糖Trehalose	+	+
硝酸盐还原Nitrate reduction	-	-	葡萄糖Glucose	+	+
棉子糖Raffinose	+	+	D-果糖D-fructose	+	-
蔗糖Sucrose	+	+	L-鼠李糖L-rhamnose	-	+
木聚糖Xylan	+	+	松三糖Melezitose	+	+
乳糖Lactose	+	+	密二糖Melibiose	+	+
菊糖Inulin	+	+	壳聚糖Chitosan	-	-
阿拉伯糖Arabinose	+	-	V-P试验V-P test	-	-

图4 葡萄糖标准曲线

Fig.4 Glucose standard curve

表2 沙福芽孢杆菌和伊朗纤维单胞菌纤维素酶的活力

Table 2 Enzyme activities of cellulases from B. safensis and C. iranensis （n=3， U·mL-1）

编号

Number

菌种

Strains

内切葡聚糖酶

Endoglucanase

外切葡聚糖酶

Exoglucanase

β-葡萄糖苷酶

β-glucosidase

伊朗纤维单胞菌C. iranensis

0.202±0.003

0.434±0.001

0.131±0.002

图5 沙福芽孢杆菌和伊朗纤维单胞菌的差异代谢物红色：伊朗纤维单胞菌相对沙福芽孢杆菌显著上调的代谢物；绿色：伊朗纤维单胞菌相对沙福芽孢杆菌显著下调的代谢物；灰色：两株菌无显著差异的代谢物；VIP：变量重要性投影；FC：差异倍数。Red： Metabolites significantly upregulated by C. iranensis relative to B. safensis； Green： Metabolites significantly downregulated by C. iranensis relative to B. safensis； Gray： No significant differences in metabolites between the two strains； VIP： Variable importance in the projection； FC： Fold change. VIP value>1； P<0.05； |log2FC|>3.

Fig.5 Differential metabolites between B. safensis and C. iranensis

图6 沙福芽孢杆菌和伊朗纤维单胞菌降解滤纸后差异显著重要代谢物热图

Fig.6 Thermogram of significant differences in important metabolites after degradation of filter paper by B. safensis and C. iranensis

图7 沙福芽孢杆菌和伊朗纤维单胞菌降解滤纸后代谢物富集分析横坐标表示每个通路对应的富集因子，纵坐标为通路名称（按照P值排序），点的颜色为P值大小，越红表示富集越显著。点的大小代表富集到差异代谢物的个数。The abscissa represents the rich factor to each pathway， the ordinate is the pathway name （sorted by P value）， and the color of the point is the P-value， and the redder the point is， the more significant the enrichment. The size of the dot represents the number of differential metabolites enriched.

Fig.7 Enrichment analysis of metabolites after degradation of filter paper by B. safensis and C. iranensis

表3 秸秆液态发酵14 d后纤维含量变化

Table 3 Changes in fiber content of straw after 14 days of liquid fermentation（n=3， % DM）

处理 Treatment	失重率 Weight loss rate	粗纤维 Crude fibre	中性洗涤纤维 Neutral detergent fibre	酸性洗涤纤维 Acid detergent fibre	半纤维素 Hemicellulose	可溶性碳水化合物 Water soluble carbohydrate
对照Control	29.18±0.91b	45.34±0.84a	69.50±0.45a	51.41±0.43a	26.81±0.03a	0.99±0.01a
沙福芽孢杆菌B. safensis	32.30±0.52b	45.68±0.79a	70.91±0.31a	48.80±0.30b	19.90±0.52b	0.11±0.03b
伊朗纤维单胞菌C. iranensis	43.12±0.19a	36.72±0.65b	52.95±0.45b	35.43±0.34b	17.53±0.49b	0.18±0.01b
P值P value	<0.01	0.03	0.02	<0.01	<0.01	<0.01

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