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Volume 42 Issue 6
Dec.  2023
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Article Navigation >  CARSOLOGICA SINICA  > 2023  >  42(6): 1202-1212
CHANG Kaiyun, WANG Zhongcheng, WEI Xiaomeng, LIU Qiumei, ZHAO Jin, ZHAO Jie, HE Xunyang. Screening and isolation of carbonic anhydrase-producing microorganisms from rocky karst habitats[J]. CARSOLOGICA SINICA, 2023, 42(6): 1202-1212. doi: 10.11932/karst20230606
Citation: CHANG Kaiyun, WANG Zhongcheng, WEI Xiaomeng, LIU Qiumei, ZHAO Jin, ZHAO Jie, HE Xunyang. Screening and isolation of carbonic anhydrase-producing microorganisms from rocky karst habitats[J]. CARSOLOGICA SINICA, 2023, 42(6): 1202-1212. doi: 10.11932/karst20230606
shu

Screening and isolation of carbonic anhydrase-producing microorganisms from rocky karst habitats

doi: 10.11932/karst20230606
  • 1.

    College of Forestry, Central South University of Forestry & Technology, Changsha, Hunan 410004, China

  • 2.

    Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan 410125, China

  • 3.

    College of Natural Resources and Environment, Northwest A&F University, Xianyang, Shaanxi 712100, China

  • Received Date: 2022-11-04
    Available Online: 2023-12-28
    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.

     

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