Screening and isolation of carbonic anhydrase-producing microorganisms from rocky karst habitats
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摘要: 喀斯特地区存在丰富的碳酸盐岩，是地球上最重要的碳库。碳酸酐酶(Carbonic anhydrase, CA)是地球上催化反应速率最快的几种酶之一，通过催化CO2的水合反应，不仅可以促进碳酸盐岩的风化，还可以通过吸收大气中的CO2来固定碳源。本研究目的是遴选喀斯特极度退化生境高CA活性菌株/菌群，探讨其用于喀斯特生态修复的可行性。利用碳酸钙培养基从喀斯特石质生境中分离筛选出产CA的菌株，并进行形态学观察、生理生化鉴定和分子生物学鉴定。通过CA活性的测定确定高CA活性的菌株，并比较单菌与多菌株组合群落的CA活性差异。利用碳酸钙培养基共分离得到产CA菌株6株，分别为耐药黄杆菌（Flavobacterium resistens）、食酸代尔夫特菌（Delftia acidovorans）、嗜根寡养单胞菌（Stenotrophomonas rhizophila）、食油假单胞菌（Pseudomonas oleovorans）、洞穴农杆菌（Agrobacterium cavarae）、白色杆菌（Bacillus albus），其中嗜根寡养单胞菌的CA活性最高，且其与其它菌株组合后CA活性均降低，说明产CA微生物的竞争作用可能大于协同作用，且在极度寡营养环境中，该嗜根寡养单胞菌具有较大的应用潜力。Abstract:
In China, the total area of rocky desertification covers 1.01×107 hm2, accounting for 22.3% of the karst area. Nowadays, rocky desertification has become one of the three major land degradation problems in our country, which has seriously affected our economic development and environmental governance. Naturally, rock weathering in the karst area is so long that it takes from a few decades to a hundred years to form a centimeter think of red soil. Because the karst area is rich in calcium and barren in soil, ecological restoration of this area is urgent in both rocky desertification control and karst carbon sink. Carbonic Anhydrase (CA), one of the fastest catalyzing enzymes, can promote the weathering of carbonate rocks by accelerating the hydration reaction of CO2, thus providing soil matrices for plant colonization. At the same time, CA can also be used to absorb CO2 in the atmosphere to fix the carbon source, thereby participating in the carbon cycle process of karst dynamic system. The CA-producing microorganisms are considered to have good application prospects in the restoration of degraded karst habitats. However, for the target strains isolated from favorable habitat, their weak adaptability to the extreme environment may constrain them from exerting their expected effects. In order to select the strain with high CA activity in the extremely degraded karst habitat, CA-producing strains were isolated from the samples of severely degraded rocky karst habitats in this study. The study area is located in Hechi City, Guangxi Zhuang (23°41'–25°37' N, 106°34'–109°09' E). It has a subtropical monsoon climate, with an average annual temperature of 16.9–21.5 ℃. The area is typically developed with karst, scattered with thin soil and highly exposed with rocks. From September to October 2021, four different habitat samples were collected, including lichen in the area with extreme rocky desertification weathered materials under moss, soil under moss, and weathered materials under moss in karst native forest. The source of isolation was a mixture of the four different habitat samples, 1g of each. CA-producing strains were isolated and screened from karst rocky habitats by inoculating suspension of isolation source in calcium carbonate medium with a sprayer. The field emission scanning electron microscope was used for morphological identification of the strain. The physiological and biochemical characteristics of the strain were confirmed by carbohydrate decomposition test, V-P test, methyl red test, citrate utilization test, nitrate reduction test, starch hydrolysis test and contact enzyme test. Strains were identified by 16S rRNA sequence analysis. The CA activity of single strain and mixed strain was determined by electrode method. The sequence alignment was performed in the National Center for Biotechnology Information Database. In MEGA6, Neighbor-Joining method is used to construct the phylogenetic tree. SPSS software was used to analyze whether there were significant differences in CA activity among different strains/microflora. The results show that six CA-producing strains were isolated by calcium carbonate medium, namely, Flavobacterium resistens, Delftia acidovorans, Stenotrophomonas rhizophila, Pseudomonas oleovorans, Agrobacterium cavarae and Bacillus albus. The CA activity of Stenotrophomonas rhizophila was the highest, up to 4.27 U·mL−1·OD600−1, while that of Bacillus albus was only 0.46 U·mL−1·OD600−1, which indicated that although CA commonly existed in prokaryotes, the activity of different species of bacteria varied greatly. Mixed culturing of Stenotrophomonas rhizophila whose CA activity is high with other strains led to a decrease in CA activity, indicating the great potential application of Stenotrophomonas rhizophila in the extremely oligotrophic environment. The results provide a new bacterial source for ecological restoration of extremely degraded karst habitats. However, all the CA activity decreased when the isolated Stenotrophomonas rhizophila strain N6 with high CA activity is mixed with other 5 strains. Because of the complex living environment, the competition of indigenous microbial community will reduce the survival efficiency of the added microorganisms, make it difficult to form a stable community structure, and then reduce the repair effect. In restoration of degraded karst habitat, whether there is a kind of dominant bacteria that is symbiotic with Stenotrophomonas rhizophila N6 to jointly promote carbonate dissolution is a research focus. Therefore, future studies should identify the core species that drive the restoration of degraded habitats, and explore the relationship between the isolated and culturable CA dominant bacteria and the core species. These study focuses will promote the research and development of biological fertilizer for the rapid restoration of degraded karst habitats. -
Key words:
- karst /
- carbonic anhydrase /
- carbon cycle /
- extremely degraded habitat /
- rocky habitat /
- ecological restoration
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图 2 不同菌种在LB固体培养基上的形态比较
Figure 2. Comparison of different bacteria species on the LB solid culture medium
图 5 不同菌种及菌群的碳酸酐酶活性
Figure 5. CA activity of different bacteria species and microbial communities
表 1 培养基的成分
Table 1. Composition of the culture medium
培养基名称 成分 碳酸钙琼脂培养基 CaCO3 50 g、ZnSO4 1 μmol、琼脂15 g、蒸馏水1 000 mL LB培养基 胰蛋白胨10 g、酵母提取物5 g、NaCl 5 g、葡萄糖1 g、蒸馏水1 000 mL 葡萄糖发酵培养基 蛋白胨1 g、NaCl 0.5 g、0.2%溴百里香酚兰1.2 mL、葡萄糖1 g、蒸馏水100 mL V-P反应培养基 蛋白胨1.5 g、葡萄糖1.5 g、K2HPO4 1.5 g、蒸馏水300 mL 固体柠檬酸盐培养基 柠檬酸钠1 g、硫酸镁0.2 g、NaCl 5 g、NH4H2PO4 1 g、K2HPO4 1 g、琼脂20 g、
1%溴麝香草酚蓝酒精溶液10 mL、蒸馏水1 000 mL硝酸盐还原培养基 硝酸钾0.2 g、蛋白胨5 g、蒸馏水1 000 mL 淀粉水解培养基 牛肉膏0.5 g、蛋白胨1 g、NaCl 0.5 g、可溶性淀粉0.2 g、琼脂2 g、蒸馏水100 mL 
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表 2 不同菌种在LB固体培养基上的形态比较
Table 2. Comparison of different bacteria species on the LB solid culture medium
样品编号 菌落形态特征 菌落颜色 N2 表面光滑，边缘整齐 黄色 N4 表面光滑，中央凸起周围扁平，边缘不规则 白色 N6 表面光滑，边缘整齐 黄色 N10 表面光滑，边缘整齐 白色 N11 表面光滑饱满，边缘整齐 白色 N13 表面光滑，蜡状，边缘不规则 白色
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表 3 菌株的生理生化特征
Table 3. Physiological and biochemical characteristics of strains
样品编号 N2 N4 N6 N10 N11 N13 糖类分解 + + + − + + V-P + + + + + + 甲基红 − − + − − + 硝酸盐还原 + + − + − − 淀粉水解 − − + − − + 柠檬酸盐利用 − + − − − − 接触酶 + + + − + −
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