Article(id=1226855197956288604, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226855188863038235, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250116, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1739894400000, receivedDateStr=2025-02-19, revisedDate=null, revisedDateStr=null, acceptedDate=1744041600000, acceptedDateStr=2025-04-08, onlineDate=1770434673060, onlineDateStr=2026-02-07, pubDate=1748966400000, pubDateStr=2025-06-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770434673060, onlineIssueDateStr=2026-02-07, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770434673060, creator=13701087609, updateTime=1770434673060, updator=13701087609, issue=Issue{id=1226855188863038235, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='6', pageStart='2321', pageEnd='2769', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1770434670891, creator=13701087609, updateTime=1770435273893, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226857718103851267, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226855188863038235, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226857718103851268, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226855188863038235, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2642, endPage=2654, ext={EN=ArticleExt(id=1226855198233112695, articleId=1226855197956288604, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=CO2-fixing microbial strains in the grassland soil of the Loess Plateau: characteristics and CO2 fixation conditions, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To screen CO2-fixing microbial strains in grassland soil, clarify their physiological and biochemical characteristics, and determine their optimal CO2 fixation conditions, providing a theoretical basis for understanding the mechanisms of soil CO2 fixation and enhancing grassland soil carbon sink. [Methods] The carbon-free solid medium and the dilution plating method were employed to isolate CO2-fixing microbial strains from grassland soil in the Loess Plateau. The strains were identified via 16S rRNA gene sequencing. Physiological and biochemical characteristics of the strains were determined by Gram staining and starch hydrolysis, indole production, methyl red, and lactic acid fermentation tests. Culture experiments with carbon-free liquid medium and sterilized soil were carried out to assess the CO2 fixation efficiency under varying temperatures (20-40 ℃), soil moisture levels (3.00%-27.00%), and the presence of electron donors (NaNO2 and Na2S). [Results] Nine bacterial strains with effective CO2 fixation were isolated, belonging to Bacillus, Streptomyces, Sinorhizobium, Agrobacterium, Enterobacter, Brevundimonas, and Prolinoborus. Among them, strains A4 (Agrobacterium) and A7 (Brevundimonas) exhibited the highest CO2 fixation efficiency (P<0.05), both being Gram-positive. Optimal conditions for CO2 fixation of the nine strains were 25-30 ℃ and soil moisture of 3.00%, 15.00%, or 27.00% (P<0.05). Electron donor supplementation enhanced CO2 fixation efficiency, and the enhancement effect followed the order: single addition of NaNO2>single addition of Na2S>co-addition of NaNO2+Na2S (P<0.05). [Conclusion] This study identified the dominant CO2-fixing microbial strains and the conditions suitable for CO2 fixation, providing a theoretical basis and technical support for enhancing the carbon sink of the terrestrial ecosystem on the Loess Plateau and mitigating the greenhouse effect.

, correspAuthors=Yimei HUANG, authorNote=null, correspAuthorsNote=
*E-mail:
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【目的】 筛选并明确草地土壤固碳微生物菌株的生理生化特性及其最适固碳条件,为深入认识土壤固碳微生物的固碳机制、提升草地土壤碳汇能力提供理论依据。【方法】 采用无碳源固体培养基和稀释涂布法,从黄土高原草地土壤中分离筛选可培养的固碳微生物菌株,并借助16S rRNA基因鉴定物种。依据革兰氏染色、淀粉水解、吲哚、甲基红和乳酸发酵试验确定其生理生化特性。利用无碳源液体培养基及灭菌土壤的培养试验,确定温度(20-40 ℃)、水分(3.00%- 27.00%),以及亚硝酸钠(NaNO2)和硫化钠(Na2S)电子供体的存在对固碳微生物固碳效率的影响。【结果】 共分离出9株固定CO2效果良好的细菌,分属芽孢杆菌属、链霉菌属、剑菌属、农杆菌属、肠杆菌属、短波单胞菌属和噬脯氨酸菌属。其中,属于农杆菌属的A4和属于短波单胞菌属的A7固碳效率最高(P<0.05),均为革兰氏阳性菌。这9株细菌在25-30 ℃,以及土壤水分3.00%、15.00%和27.00%的条件下,固碳效率较高(P<0.05)。添加电子供体可提升菌株固碳效率,提升效果依次为:单独添加NaNO2>单独添加Na2S>共同添加NaNO2+Na2S (P<0.05)。【结论】 本研究确定了优势固碳微生物菌株以及适宜固定CO2的条件,为提升黄土高原陆地生态系统碳汇、减缓温室效应提供了理论依据和技术支持。

, correspAuthors=黄懿梅, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=ZJ1I2DvZMnFKWTsTUeDA1w==, magXml=lygAIVYkJPQQewO2PS1nVg==, pdfUrl=null, pdf=qBfJBXc2eTZ1Tgs43Fa8LQ==, pdfFileSize=1386527, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=MovsxwqNn55LV07DiwZcrA==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=67IbUmAOXXCfcuM3MzISog==, mapNumber=null, authorCompany=null, fund=null, authors=

作者贡献声明

雷发丹:数据收集和处理,文章撰写和修改;黄倩:研究构思和设计;陆晓钰:协助固碳条件探究试验;曹雨晴:协助筛菌工作;魏宾:协助菌株特性测定;徐嘉宜:协助筛菌工作;陈芳:协助指标测定;王宝荣:参与文章讨论;黄懿梅:项目管理,经费支持,文章审阅。

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Different lowercase letters: The significant difference between different treatments of the same strain (P<0.05); Different capital letters: The significant differences among different strains under the same treatment conditions (P<0.05)., figureFileSmall=JD2ctbZFDVcRfvyNDW9Xdg==, figureFileBig=hJh5e5Hzgl1l2f4ZolZKfA==, tableContent=null), ArticleFig(id=1227680964231492573, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226855197956288604, language=CN, label=图3, caption=不同水分对CO2 固定菌株固碳效率的影响, figureFileSmall=JD2ctbZFDVcRfvyNDW9Xdg==, figureFileBig=hJh5e5Hzgl1l2f4ZolZKfA==, tableContent=null), ArticleFig(id=1227680964374098918, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226855197956288604, language=EN, label=Figure 4, caption=Effects of different electron donors on CO2 fixation efficiency of CO2-fixing strains. 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Physiological and biochemical characteristics of strains

, figureFileSmall=null, figureFileBig=null, tableContent=
StrainsStarch hydrolysisIndole reactionMethyl red reactionLactose acid productionLactose gas production
A1--+--
A2--+++
A3--+--
A4-+---
A5-+---
A6-+---
A7+-+--
A8--+--
A9--+--
), ArticleFig(id=1227680964780945408, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226855197956288604, language=CN, label=表1, caption=

菌株的生理生化特性

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StrainsStarch hydrolysisIndole reactionMethyl red reactionLactose acid productionLactose gas production
A1--+--
A2--+++
A3--+--
A4-+---
A5-+---
A6-+---
A7+-+--
A8--+--
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黄土高原草地土壤固碳微生物菌株特性及其固碳条件
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雷发丹 1 , 黄倩 1 , 陆晓钰 1 , 曹雨晴 1 , 魏宾 1 , 徐嘉宜 1 , 陈芳 1 , 王宝荣 2 , 黄懿梅 1, *
微生物学报 | 研究报告 2025,65(6): 2642-2654
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微生物学报 | 研究报告 2025, 65(6): 2642-2654
黄土高原草地土壤固碳微生物菌株特性及其固碳条件
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雷发丹1, 黄倩1, 陆晓钰1, 曹雨晴1, 魏宾1, 徐嘉宜1, 陈芳1, 王宝荣2, 黄懿梅1, *
作者信息
  • 1.西北农林科技大学 资源环境学院,农业农村部西北旱地农业绿色低碳重点实验室,陕西 杨凌
  • 2.西北农林科技大学 草业与草原学院,陕西 杨凌
CO2-fixing microbial strains in the grassland soil of the Loess Plateau: characteristics and CO2 fixation conditions
Fadan LEI1, Qian HUANG1, Xiaoyu LU1, Yuqing CAO1, Bin WEI1, Jiayi XU1, Fang CHEN1, Baorong WANG2, Yimei HUANG1, *
Affiliations
  • 1.Key Laboratory of Low-carbon Green Agriculture in Northwestern China, Ministry of Agriculture and Rural Affairs, College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi, China
  • 2.College of Grassland Agriculture, Northwest A&F University, Yangling, Shaanxi, China
出版时间: 2025-06-04 doi: 10.13343/j.cnki.wsxb.20250116
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【目的】 筛选并明确草地土壤固碳微生物菌株的生理生化特性及其最适固碳条件,为深入认识土壤固碳微生物的固碳机制、提升草地土壤碳汇能力提供理论依据。【方法】 采用无碳源固体培养基和稀释涂布法,从黄土高原草地土壤中分离筛选可培养的固碳微生物菌株,并借助16S rRNA基因鉴定物种。依据革兰氏染色、淀粉水解、吲哚、甲基红和乳酸发酵试验确定其生理生化特性。利用无碳源液体培养基及灭菌土壤的培养试验,确定温度(20-40 ℃)、水分(3.00%- 27.00%),以及亚硝酸钠(NaNO2)和硫化钠(Na2S)电子供体的存在对固碳微生物固碳效率的影响。【结果】 共分离出9株固定CO2效果良好的细菌,分属芽孢杆菌属、链霉菌属、剑菌属、农杆菌属、肠杆菌属、短波单胞菌属和噬脯氨酸菌属。其中,属于农杆菌属的A4和属于短波单胞菌属的A7固碳效率最高(P<0.05),均为革兰氏阳性菌。这9株细菌在25-30 ℃,以及土壤水分3.00%、15.00%和27.00%的条件下,固碳效率较高(P<0.05)。添加电子供体可提升菌株固碳效率,提升效果依次为:单独添加NaNO2>单独添加Na2S>共同添加NaNO2+Na2S (P<0.05)。【结论】 本研究确定了优势固碳微生物菌株以及适宜固定CO2的条件,为提升黄土高原陆地生态系统碳汇、减缓温室效应提供了理论依据和技术支持。

黄土高原草地  /  固碳微生物菌株  /  固碳条件  /  固碳效率  /  电子供体

[Objective] To screen CO2-fixing microbial strains in grassland soil, clarify their physiological and biochemical characteristics, and determine their optimal CO2 fixation conditions, providing a theoretical basis for understanding the mechanisms of soil CO2 fixation and enhancing grassland soil carbon sink. [Methods] The carbon-free solid medium and the dilution plating method were employed to isolate CO2-fixing microbial strains from grassland soil in the Loess Plateau. The strains were identified via 16S rRNA gene sequencing. Physiological and biochemical characteristics of the strains were determined by Gram staining and starch hydrolysis, indole production, methyl red, and lactic acid fermentation tests. Culture experiments with carbon-free liquid medium and sterilized soil were carried out to assess the CO2 fixation efficiency under varying temperatures (20-40 ℃), soil moisture levels (3.00%-27.00%), and the presence of electron donors (NaNO2 and Na2S). [Results] Nine bacterial strains with effective CO2 fixation were isolated, belonging to Bacillus, Streptomyces, Sinorhizobium, Agrobacterium, Enterobacter, Brevundimonas, and Prolinoborus. Among them, strains A4 (Agrobacterium) and A7 (Brevundimonas) exhibited the highest CO2 fixation efficiency (P<0.05), both being Gram-positive. Optimal conditions for CO2 fixation of the nine strains were 25-30 ℃ and soil moisture of 3.00%, 15.00%, or 27.00% (P<0.05). Electron donor supplementation enhanced CO2 fixation efficiency, and the enhancement effect followed the order: single addition of NaNO2>single addition of Na2S>co-addition of NaNO2+Na2S (P<0.05). [Conclusion] This study identified the dominant CO2-fixing microbial strains and the conditions suitable for CO2 fixation, providing a theoretical basis and technical support for enhancing the carbon sink of the terrestrial ecosystem on the Loess Plateau and mitigating the greenhouse effect.

Loess Plateau grassland  /  CO2-fixing microbial strains  /  CO2 fixation conditions  /  CO2 fixation efficiency  /  electron donor
雷发丹, 黄倩, 陆晓钰, 曹雨晴, 魏宾, 徐嘉宜, 陈芳, 王宝荣, 黄懿梅. 黄土高原草地土壤固碳微生物菌株特性及其固碳条件. 微生物学报, 2025 , 65 (6) : 2642 -2654 . DOI: 10.13343/j.cnki.wsxb.20250116
Fadan LEI, Qian HUANG, Xiaoyu LU, Yuqing CAO, Bin WEI, Jiayi XU, Fang CHEN, Baorong WANG, Yimei HUANG. CO2-fixing microbial strains in the grassland soil of the Loess Plateau: characteristics and CO2 fixation conditions[J]. Acta Microbiologica Sinica, 2025 , 65 (6) : 2642 -2654 . DOI: 10.13343/j.cnki.wsxb.20250116
作为陆地生态系统最主要的碳库,土壤有机碳的微小波动就能显著改变大气CO2浓度,影响全球碳平衡[1]。土壤微生物碳泵理论的提出,凸显了微生物在土壤碳转化与储存过程中的核心地位[2]。固碳微生物作为这一过程的特殊执行者,能够同化CO2贡献有机碳库的形成,其包括光能自养型和化能自养型微生物,以及部分异养微生物[3]。自养微生物每年为陆地有机碳库(1 550 pg)贡献4.90 pg C[4],其固碳潜力不可忽视。目前已知的固碳微生物及其固碳潜力研究,大多采用稳定同位素标记耦合宏基因组测序技术进行,虽能全面明确群落和固碳潜力,但设备和操作要求高,且无法获得功能菌株[5-6]。最新的高通量菌株筛选技术能够快速处理大量样本、挖掘稀有固碳微生物并分析基因信息,但存在设备成本高、对人员要求高、田间应用受限及鉴定不够精确等问题[7]。尽管传统的分离培养法受培养基成分和培养条件限制,可成功分离鉴定的固碳微生物种类有限[8],但可以获得可挖掘的菌株,且结合目前的PCR测序技术,可准确鉴定其类群[9],有助于挖掘土壤固碳微生物菌株资源和深入认识其生理特性。
温度、降水、生态系统与土壤类型、土壤有机质及养分含量等气候与环境因素均会影响固碳微生物群落及功能[10-13]。能量和电子源也是影响微生物固定CO2的关键因素。在特定电子供体作用下,固碳微生物的固碳效率可提升数倍以上[14-16]。明确固碳菌株的固碳效率、电子供体偏好性及环境适应性,能为后续菌株功能改良及应用提供重要理论依据[17-19]。黄土高原气候条件恶劣,土壤侵蚀现象严重,土质贫瘠,有机质含量处于较低水平。在各类土地利用方式中,草地对于维持该地区生态系统的稳定与可持续性具有显著优势[20]。草地覆盖了约1/3的黄土高原区域[21],该区域草地土壤固碳量占草地总固碳量的59.53%[22],其中固碳微生物对有机碳的贡献率可达0.03%[23]。然而,可培养固碳菌株的种类、特性、功能以及适宜的固碳条件仍不明确。
本研究聚焦黄土高原草地,旨在从其土壤中分离鉴定CO2固定菌株,通过16S rRNA基因测序分析、形态观察以及生理生化反应测定,全面鉴定菌株。同时,探究不同温度、不同水分,以及亚硝酸钠(NaNO2)和硫化钠(Na2S) 2种电子供体单独和共同作用对固碳菌群生长情况和固碳效率的影响,以期确定优势固碳微生物菌株以及适宜固定CO2的条件,为提升黄土高原陆地生态系统碳汇、减缓温室效应提供理论依据与技术支持。
2019年8月,在黄土高原安塞区(36°51′30′′N,109°19′23′′E)选择3个坡向和海拔相近(100 m×100 m,间隔100 m)的样地。在每个样地沿“S”型布设7个样点,采集0-5 cm表层土壤,均匀混合为1个代表样品。草地主要植被包括狗尾草[Setaria viridis (L.) P. Beauv.]、草木樨(Melilotus suaveolens Ledeb.)、斜茎黄芪(Astragalus laxmannii Jacq.)、地锦草(Euphorbia humifusa Willd. ex Schltdl.)、细裂叶莲蒿(Artemisia gmelinii Weber ex Stechm.)、兴安胡枝子[Lespedeza davurica (Laxm.) Schindl.]、苦马豆[Sphaerophysa salsula (Pall.) DC.]和披针叶野决明(Thermopsis lanceolata R. Br.)。3个土壤样品的理化性质并无显著差异(P<0.05),土壤呈偏碱性,pH值为8.24±0.09,含水率为(15.00±2.00)%,有机碳含量为(5.74±0.98) g/kg,全氮含量为(0.64±0.07) g/kg。
本研究中使用的培养基包括:无碳源培养基[24]、淀粉培养基[22]、葡萄糖蛋白胨水培养基[25]、蛋白胨水培养基[22]、乳酸发酵培养基[25]
将10.00 g土壤样品充分混合于90 mL无菌水中,30 ℃、150 r/min振荡20 min后制成土壤悬浊液。将悬浊液进行浓度稀释(无菌操作),并吸取10-5、10-6、10-7稀释浓度下的样品悬浊液1 mL均匀涂布于无碳源固体培养基(凝结物质:琼脂)上,28 ℃倒置培养10-14 d (光12 h+暗12 h)。待菌株长出后,进行多次划线分离纯化,挑取单菌落于4 ℃保存备用。
将挑取的单菌落接种于无碳源液体培养基中,30 ℃、150 r/min培养48 h。培养结束后吸取菌液至无菌5 mL离心管中,25 ℃、3 000 r/min离心10 min,收集菌体,储存在4 ℃冰箱中。按照Ezup柱式细菌基因组DNA抽提试剂盒的操作步骤,提取不同菌株的基因组DNA,置于-20 ℃冰箱中保存备用。细菌物种鉴定由上海美吉生物医药科技有限公司(Shanghai Majorbio Bio-pharm Technology Co., Ltd.)完成。采用引物27F (5′-AGAGTTTGATC CTGGCTCAG-3′)和1492R (5′-GGTTACCTTGT TACGACTT-3′)进行PCR扩增。PCR反应体系:10×Ex Taq buffer 2.00 μL,Ex Taq (5 U/μL) 0.20 μL,dNTP Mix (2.50 mmol/L) 1.60 μL,上、下游引物(10 μmol/L)各1 μL,DNA 0.50 μL,ddH2O 13.70 μL。PCR反应条件:95 ℃预变性5 min;95 ℃变性30 s,56 ℃退火30 s,72 ℃延伸90 s,共25个循环;72 ℃终延伸10 min。扩增完成后,以120 V电压进行1%琼脂糖凝胶电泳30 min。扩增的序列使用ABI 3730XL测序仪进行一代双末端测序,获得ABI测序峰图文件。将正常峰的菌株碱基序列与NCBI GenBank核酸序列数据库进行比对,获取近源物种信息,并构建系统发育树[原始数据存储在国家微生物科学数据中心(http://nmdc.cn),编号为NMDCN0007OUI、NMDCN0007OUJ、NMDCN0007OUK、NMDCN0007OUL、NMD CN0007OUM、NMDCN0007OUN、NMDCN0007OUO、NMDCN0007OUP、NMDCN0007OUQ、NMDCN0007OUR]。
对分离筛选到的菌株进行菌落特征、生长情况的观察以及革兰氏染色、镜检[26]
将分离筛选到的菌株进行淀粉水解试验、吲哚试验、甲基红试验以及乳酸发酵试验,观察并记录生理生化反应结果[27]
将40 mL无碳源液体培养基装入100 mL三角瓶,灭菌后加入1 mL菌液,用封口膜封口,置于摇床(28 ℃、100 r/min)振荡培养72 h活化菌株。
采用单时间点终止培养法探讨温度对固碳微生物的影响,选择20-40 ℃作为温度探究区间,此范围覆盖了该地区一年中大部分时段的土壤温度变化,能够有效考察固碳微生物在黄土高原常规温度条件下的生长与固碳表现[28]。设计5个温度梯度:20、25、30、35、40 ℃,吸取活化后的菌株1 mL接种于无碳源液体培养基中在不同温度梯度下置于摇床(100 r/min)振荡培养72 h。
采用单时间点终止培养法探讨电子供体对固碳微生物的影响,设计NaNO2和Na2S这2种处理,采用0 g/L和1.00 g/L两个浓度水平。将各菌株接种于无碳源液体培养基中,分别加入对应浓度水平的电子供体后,置于摇床(100 r/min)振荡培养72 h。
在水分条件设置上,基于黄土高原降水不稳定,土壤水分在不同季节和年份呈现出较大幅度变化的特点[28],以该地区草地土壤自然水分(15.00%)为基准,增设增减30.00%、增减60.00%以及增减80.00%的梯度,系统研究固碳微生物在接近自然状态下不同水分胁迫与充裕条件下的固碳特性。土壤水分设计7个梯度:3.00%、6.00%、10.50%、15.00%、19.50%、24.00%和27.00%含水率。称取安塞草地表层土壤13.00 g于50 mL烧杯中,铺成1 cm厚的薄层,封口并扎紧,并使用高压蒸汽灭菌锅(121 ℃、20 min)将土壤灭菌6次并烘干。在不同水分梯度下添加菌液1 mL,于28 ℃下培养30 d。培养期间,间隔3 d补充灭菌蒸馏水,以维持土壤含水率。
土壤含水率采用称重法测定,土壤pH通过土水质量体积比1:2.5使用pH计测定,土壤有机碳(soil organic carbon, SOC)采用重铬酸钾外加热法[23]测定,土壤总氮采用凯氏定氮法[23]测定,菌液总有机碳(total organic carbon, TOC)值通过总有机碳分析仪(岛津公司)进行测定,测定时需提前用浓盐酸将待测菌液调至pH 4.0左右,以消除无机碳对测定结果的影响。
不同温度梯度和电子供体条件下菌株固碳效率TOC (mg/L)的计算如公式(1)所示。
TOC=TOC72 h
式中:TOC72 h (mg/L)为液体培养基培养72 h后的TOC值。
不同水分梯度条件下菌株固碳效率ΔSOC [g/(kg·d)]的计算如公式(2)所示。
ΔSOC=(SOC30 d-SOC0 d)/30
式中:SOC30 d (g/kg)为添加菌株后培养第30天时的土壤有机碳含量,SOC0 d (g/kg)为添加菌株后培养第0天时的土壤有机碳含量,30为培养天数。
所有数据均以平均值±标准偏差的形式表示。使用SPSS软件对所有样品进行单因素方差分析以及最小显著差异多重比较,以显著性水平(P<0.05)分析组间数据的差异。数据绘图在Origin 2018中完成。
从草地土壤中分离得到了24株菌株,测定第1、2、5、6、7、11、13、14、16和22株菌在1 400 bp附近出现的PCR产物条带清晰,未观察到明显的非特异性条带或拖尾现象,且ABI测序峰图为正常峰,证实以上10株菌株为纯菌株。将10株纯菌株的碱基序列输入NCBI GenBank核酸序列数据库进行比对,结果显示:1号菌株与恶臭假单胞菌(Pseudomonas putida) M9相似度为98.32%,属于假单胞菌属;2号菌株与蜡样芽孢杆菌(Bacillus cereus) LPBS12相似度为91.80%,属于芽孢杆菌属;5号菌株与链霉菌属(Streptomyces)FXJ3.004相似度为98.94%,属于链霉菌属;6号菌株与草木栖剑菌(Sinorhizobium meliloti)TSA1相似度为98.75%,属于剑菌属;7号菌株与农杆菌属(Agrobacterium) CRRI-74 SB13A相似度为97.68%,属于农杆菌属;11号菌株与链霉菌属(Streptomyces) IRB27B相似度为96.84%,属于链霉菌属;13号菌株与华西医院肠杆菌(Enterobacter huaxiensis) N7相似度为98.06%,属于肠杆菌属;14号菌株与泡囊短波单胞菌(Brevundimonas vesicularis)TK026相似度为95.55%,属于短波单胞菌属;16号菌株与农杆菌属(Agrobacterium) S3D11相似度为98.96%,属于农杆菌属;22号菌株与小束噬脯氨酸菌(Prolinoborus fasciculus)L61相似度为97.56%,属于噬脯氨酸菌属(图1)。其中芽孢杆菌属菌株隶属于厚壁菌门,链霉菌属菌株隶属于放线菌门,其余菌株均隶属于变形菌门。
对以上10株纯菌株进行初步筛选,发现1号菌株在后续预培养过程中对试验条件极为敏感,培养基成分或培养条件的轻微波动都会导致其生长停滞。基于以上原因,为了确保试验过程高效稳定、结果准确可靠,后续研究中仅选取了其余9株纯菌株作为研究对象,并对这9株纯菌株重新编号。原2、5、6、7、11、13、14、16、22号菌株分别重新编号为A1、A2、A3、A4、A5、A6、A7、A8、A9。
菌落形态均为黏着突起,有球状、棒状、链状,部分细胞还含有孢子。颜色各异:A1、A3、A7、A8为白色;A4、A6、A9呈黄色;A2为红色。链霉菌A2和A5有明显刺激性气味。其中,菌株A3、A4、A7、A8呈紫红色,为革兰氏阳性菌;其余菌株呈红色,为革兰氏阴性菌。
九株纯菌株的生理生化反应情况见表1。A7的淀粉水解反应呈阳性;A4、A5、A6的吲哚反应呈阳性;A1、A2、A3、A7、A8、A9的甲基红反应呈阳性;A2具备产酸产气能力。
可培养的CO2固定菌株在不同温度下均表现出一定的固碳能力,且对温度变化的响应规律一致:在25-30 ℃时固碳效率达到峰值,温度升高或降低均会导致固碳效率下降(图2P<0.05)。在25 ℃和30 ℃时,菌株A4的固碳效率在所有受试菌株中最高,分别为4.04 mg/L和3.98 mg/L,而其他菌株在这2个温度下的平均固碳效率分别为3.20 mg/L和3.16 mg/L。与20、35、40 ℃相比,菌株A4在25 ℃时的TOC值分别显著增加了77.30%、71.50%和95.90%;在30 ℃时,其TOC值相比上述温度也分别显著提升了74.70%、69.00%和93.00%。
可培养的CO2固定菌株对土壤水分变化的响应规律一致(图3P<0.05)。在自然水分(15.00%)及增减80.00% (27.00%和3.00%)的水分条件下,菌株固碳效率最高。其中,菌株A4在3.00%和27.00%水分条件下的固碳量显著高于其他水分条件,分别达到0.15 g/(kg·d)和0.11 g/(kg·d)。在15.00%水分条件下,各菌株固碳量无显著差异。在10.50%水分条件下,菌株A8固碳量最高,为0.03 g/(kg·d);19.50%水分条件下,菌株A2固碳量最高,为0.02 g/(kg·d);在6.00%水分条件下,各菌株固碳量无显著差异;在24.00%水分条件下,菌株A4和A5固碳量最高,均为0.01 g/(kg·d)。在3.00%水分条件下,菌株A6和A7固碳量最低,分别为0.06 g/(kg·d)和0.05 g/(kg·d);27.00%水分条件下,菌株A6固碳量最高,达到0.19 g/(kg·d)。
同一菌株在不同电子供体处理条件下的固碳效率差异显著:单独添加NaNO2>单独添加Na2S>共同添加NaNO2+Na2S>空白对照(图4P<0.05)。在空白对照组中,菌株A4和A7的固碳量最高,TOC值分别为4.22 mg/L和4.08 mg/L,其余菌株的平均固碳量为3.42 mg/L。添加电子供体后,固碳效率显著提升,其中提升最显著的是菌株A4,其次是A7。单独添加NaNO2时,菌株A4的TOC值达到16.10 mg/L,是空白对照组的3.82倍;菌株A7的TOC值为14.80 mg/L,是空白对照组的3.64倍。单独添加Na2S时,菌株A4的TOC值为9.28 mg/L,是空白对照组的2.20倍;菌株A7的TOC值为9.00 mg/L,是空白对照组的2.21倍。共同添加NaNO2+Na2S时,菌株A4的TOC值为7.26 mg/L,是空白对照组的1.72倍;菌株A7的TOC值为6.99 mg/L,是空白对照组的1.71倍。此外,菌株A5和A8在不同电子供体环境下的固碳量仅次于A4和A7。菌株A5在单独添加NaNO2、Na2S及共同添加NaNO2+Na2S环境下的固碳量分别为8.32、6.35、5.01 mg/L;菌株A8对应值分别为9.03、7.00、4.89 mg/L。其余5株菌株在单独添加NaNO2、单独添加Na2S和共同添加NaNO2+Na2S环境下的平均固碳量分别为6.54、4.73、4.07 mg/L。
本研究从黄土高原草地土壤中成功分离筛选出9株CO2固定菌株,这些菌株与从海水、深海热液区筛选出的化能自养菌,包括弧菌属(Vibrio)、硫膨大杆菌属(Thioclava)、大洋芽孢杆菌属(Oceanobacillus)、食烷菌属(Alcanivorax)、氢单胞菌属(Hydrogenimonas)、氧化硫单胞菌属(Sulfurimonas)及硫化螺旋菌属(Sulfurospirillum)等,分属于不同的微生物类群[14,29];与从种植花生的农田土壤中筛选出的芽孢杆菌属(Bacillus) SS105及种植竹子的根际土壤中筛选出的链霉菌属(Streptomyces) BS19属于相同菌属[9,30],但具体菌株有所不同。前人筛选出的酸硫杆状菌属(Acidithiobacillus)和食酸菌属(Acidovorax)等固碳菌株,因其独特的生理代谢特征和极端环境适应性,已广泛应用于生物浸出、生物修复等领域[31-32]。本研究的9株固碳菌株均为新菌株,能在更宽的温度和湿度范围内保持高固碳效率,且对特定电子供体的利用效率更高,为土壤碳汇提供了新的资源[33]。在无碳源培养基中,通过终态TOC值的增加,证实了菌株能够同化CO2合成有机碳[34]。尽管未测定初始TOC背景值可能存在轻微误差,但菌株间终态TOC的显著差异仍清晰地呈现出固碳效率的排序[35]。其中,A4和A7的固碳效率最高,终态TOC值分别达到4.22 mg/L和4.08 mg/L。需要注意的是,实验室中的单菌培养与土壤中环境不同,土壤中复杂的碳源可能抑制菌株固碳酶的活性[36],空间异质性也可能限制CO2的扩散[37]。因此,菌株在实际土壤中的固碳效能需要借助13C同位素标记微宇宙试验进行验证[38],并结合转录组学解析其在多碳源胁迫下的代谢调控网络[39]。未来的研究将整合固碳动力学建模、菌体-分泌碳分配量化[40]以及田间原位验证,以评估菌株在生态固碳工程中的应用潜力。
本研究筛选的可培养CO2固定菌株,在25-30 ℃区间内固碳效率达到峰值,推测该温度范围有利于提升固碳酶的活性,而温度的升高或降低则因抑制酶活性而导致固碳效率降低[41]。不同生态系统的固碳菌株最适固碳温度区间存在差异,例如武满满等[10]从海水及其沉积物中分离的固碳微生物在20-30 ℃时固碳效率较低,但温度的改变反而能提升其效率。这可能源于不同生态环境使菌株进化出了不同的适应策略[41]。黄土高原草地夏季高温期长、冬季寒冷且温差大[22,28],本研究菌株在较大温度范围内具有固碳能力,预示其在该地区具有良好的应用潜力。研究还发现,菌株在自然水分(15.00%)及极端水分(3.00%和27.00%)条件下均表现出显著的固碳效率峰值,呈现出“双向胁迫响应”特性,这可能与固碳菌维持代谢活性的多维度适应策略相关[42-43]。黄土高原降水不均,旱雨季土壤水分差异大[28],这些菌株能够适应此类极端水分变化。在自然水分条件下,微生物细胞的生理功能稳定,有利于固碳酶发挥活性[5];而在水分极低(3.00%)时,固碳菌启动应激反应,合成相容性溶质、表达特殊转运蛋白以提高固碳效率[27];在水分极高(27.00%)时,固碳菌可能调整代谢途径以应对缺氧挑战(如采用厌氧还原柠檬酸循环或乙酰辅酶A途径)[5]。此外,在15.00%和6.00%水分条件下,9株菌株的固碳效率无显著差异,这可能是因为相关代谢途径及关键酶的运行状态相同[27]
本研究表明,NaNO2和Na2S作为电子供体,可有效提升CO2固定菌的固碳效率,这与化能自养型固碳微生物以还原性无机物为能源、CO2为碳源合成生命物质时的ATP合成及无机化合物氧化过程相关[14]。黄土高原草地土壤中含有一定量的NO2-和S2-[44],该研究结果对区域固碳应用具有重要意义。在空白对照中,菌株A4和A7的固碳效率较高。添加电子供体后,A4的固碳效率提升效果显著且在各菌株中最高,A7次之。这表明A4和A7自身固碳能力强,为深入研究黄土高原草地土壤环境中微生物固碳机制提供了优质的菌株模型。单独添加NaNO2时,提升固碳效率的效果最显著,其次单独添加Na2S。这与Hu等[43]的研究相反(其研究中,Na2S对从海水中分离的非光合微生物群落固CO2效率的提升效果高于NaNO2),可能是本试验菌株对NO2-的利用能力强,有利于其在NO2-环境下繁殖及还原CO2[45]。研究表明,合适的电子供体可优化微生物的呼吸代谢途径,增强CO2固定能力[43]。单独添加Na2S时,固碳效果次之,这可能是因为S2-参与菌株代谢时,氧化过程的能量和电子传递效率较低;共同添加NaNO2和Na2S时,固碳效果反而减弱,甚至低于单独添加Na2S,这可能是NO2-和S2-存在竞争性关系,干扰了菌株细胞内电子传递链和能量代谢;在微生物代谢中,电子传递链是固碳反应能量产生的关键,NO2-和S2-竞争关键酶,影响酶正常作用和能量产生效率[46]。例如在某些固碳微生物中,细胞色素氧化酶对NO2-和S2-的亲和力不同,二者同时存在会打破酶与底物结合的平衡,影响电子传递,进而降低固碳效率[47-49]
本研究表明,温度、水分和电子供体显著影响菌株的固碳能力,且不同菌株之间的差异明显。未来在黄土高原草地退化区的生态修复中,可根据需要定制菌剂:在环境恶劣的退化区,选用A4、A7等固碳能力强、适应性好的菌株,加快生态恢复;在环境温和的退化区,选择A2等菌株,优化微生物群落,提升土壤碳汇能力。
(1) 本研究从黄土高原草地土壤中筛选鉴定出9株CO2固定菌,分属芽孢杆菌属、链霉菌属、剑菌属、农杆菌属、肠杆菌属、短波单胞菌属、噬脯氨酸菌属。其中,A4和A7的固碳效率显著高于其他菌株(P<0.05)。(2) 这些菌株在20-40 ℃均可固定CO2,25-30 ℃为最适温度。在土壤自然水分(15.00%)及增减80.00%含水率(27.00%和3.00%)的极端条件下,固碳效率最高(P<0.05)。(3) 添加电子供体能提升固碳效率,提升效果依次为:单独添加NaNO2>单独添加Na2S>共同添加NaNO2+Na2S (P<0.05)。本研究深化了对固碳微生物的认知,为增强黄土高原土壤固碳微生物活性提供了理论支撑。
作者声明绝无任何可能会影响本文所报告工作的已知经济利益或个人关系。
  • 国家自然科学基金(42477372)
  • 西北农林科技大学大学生创新创业训练计划(XN2025007089)
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doi: 10.13343/j.cnki.wsxb.20250116
  • 接收时间:2025-02-19
  • 首发时间:2026-02-07
  • 出版时间:2025-06-04
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  • 收稿日期:2025-02-19
  • 录用日期:2025-04-08
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National Natural Science Foundation of China(42477372)
国家自然科学基金(42477372)
College Students’ Innovative Entrepreneurial Training Program of Northwest A&F University(XN2025007089)
西北农林科技大学大学生创新创业训练计划(XN2025007089)
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    1.西北农林科技大学 资源环境学院,农业农村部西北旱地农业绿色低碳重点实验室,陕西 杨凌
    2.西北农林科技大学 草业与草原学院,陕西 杨凌

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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