固碳微生物菌株的分离鉴定及其固碳能力测定

利用微生物回收和固定CO2气体的生物固碳方法,成为解决"温室效应"这一重大环境问题的焦点,本研究目的是分离筛选固碳微生物菌株。利用无碳源无机培养基从活性污泥、沼液和设施土壤中分离筛选出以CO2为碳源的菌株24株,选取其中生长较快的8株菌进行cbbL基因、形态观察、生理生化反应测定和16S rDNA测序分析,将测序结果在BlAST数据库中比对,进行固碳菌株的分子鉴定,并对其菌体含量和RubisCO酶活性进行比较。选取RubisCO酶活性最高的菌株C2-8R,进行土壤施用试验,通过土壤RubisCO酶活性的测定,确定分离筛选的固碳菌的固碳能力。研究表明,可通过无碳源培养基进行固碳微生物菌株的筛选,筛选的固碳菌分别隶属于假单胞菌属和嗜甲基菌属,并可通过RubisCO酶活性来反映微生物的固碳能力。     

绿藻CO2浓缩机制的研究进展

单细胞绿藻是淡水水体中浮游植物的重要组成部分,也是淡水生态系统中主要的初级生产者,其在适应外界CO2浓度变化的过程中,细胞内形成了一种主动转移无机碳的机制－CO2浓缩机制（CO2 concentrating mechanism,CCM）。该机制能使细胞在核酮糖－2－磷酸羧化氧化酶（rubiscol)固碳位点提高CO2浓度,以增加光合作用和减少光吸收。本文综述了这种机制中的无机碳转移模型和不同环境因子（光,温度,CO2浓度和营养水平）对它的调控作用,以期促进深入开展浮游植物对大气CO2浓度升高响应的研究。     

羧酶体的组成、结构、功能和检测及其在脱氮细菌中的意义

羧酶体(Carboxysome)是一种具有CO2浓缩功能的"类细胞器",它存在于自养型脱氮细菌中,可增强细菌的自养生长能力。硝化细菌、厌氧氨氧化细菌和部分反硝化细菌都是重要的自养型脱氮细菌,探明其羧酶体的组成、结构和功能,将有助于揭示自养型脱氮菌的生长规律,进而强化生物脱氮过程。基于文献阅读和相关研究,本文对自养型细菌中羧酶体在组成、结构、功能和检测等方面的研究进展进行综述,以期为自养生物脱氮过程的深入理解和优化改进提供参考。     

赤潮藻中肋骨条藻的光合作用对海水pH和N变化的响应

为探讨赤潮发生时中肋骨条藻 (Skeletonemacoatatum)的光合作用生理变化 ,研究了不同无机氮 (N)水平上 ,海水pH值升高对其胞外碳酸酐酶 (CA)和光合生理特性的影响。海水pH从 8.2升至 8.7时 ,中肋骨条藻胞外CA被诱导 ,细胞对无机碳的亲和力 (1/Km)提高 ;在pH8 7时 ,高N条件下的胞外CA活性是低N条件下的 3倍 ,1/Km 值也提高了 80 %。单位叶绿素a的最大净光合能力 (Pam)在不同pH和N水平上没有显著差异 ;但单位细胞的最大净光合能力 (Pcm)提高了 10 0 %。这些结果表明 ,赤潮发生时 ,中肋骨条藻通过启动无机碳浓缩机制 (CCM) ,提高细胞对无机碳利用效率 ,使其在低CO2 (高pH)环境下维持光合机构正常运行 ;充足的N源有利于提高CCM的效率 ,从而提高CO2 环境下的光合固碳能力。     

微藻生物技术在碳中和的应用与展望

碳中和是指CO2"零排放",在一段时间内通过节能减排、增加碳汇等途径,抵消各类活动所产生的CO2的排放.微藻是含有叶绿素a的原生生物,可以利用太阳能通过浓缩机制(CCM)进行光合作用高效固定CO2、通过异养同化作用转化固定有机碳.微藻生物质可转化为生物燃料、生物材料及生物肥料等,实现对传统化石燃料、塑料及化肥等的替代....     

绿藻CO2浓缩机制的研究进展

单细胞绿藻是淡水水体中浮游植物的重要组成部分,也是淡水生态系统中主要的初级生产者,其在适应外界环境CO₂浓度变化的过程中,细胞内形成了一种主动转移无机碳的机制———CO₂浓缩机制(CO₂concentrating mechanism,CCM).该机制能使细胞在核酮糖2磷酸羧化氧化酶(rubisco)固碳位点提高CO₂浓度,以增加光合作用和减少光呼吸.本文综述了这种机制中的无机碳转移模型和不同环境因子(光、温度、CO₂浓度和营养水平)对它的调控作用,以期促进深入开展浮游植物对大气CO₂浓度升高响应的研究.     

非光合微生物菌群好氧固定CO2的研究

微生物固定CO2在环境、资源方面具有重要意义.然而,通常具有较高固碳能力的光合细菌和氢-氧化细菌由于需要光照/严格厌氧和供氢,限制了其在反应器或土壤中的应用.本研究通过生物技术手段从海水及其沉积物中选育到在普通好氧条件下具有固碳能力的非光合微生物菌群,并通过电子供体和无机碳源结构的优化,显著提高了其对无机碳的同化能力,好氧条件下固碳菌液的最高无机碳同化效率可达110 mgCO2 / L·d,为实现普通环境条件下的微生物固碳奠定了基础.同时,通过分子生物学手段研究了不同环境条件下固碳微生物菌群的群落结构,以期为进一步优化固碳微生物群落结构,提升固碳效率提供理论依据.结果发现在不同培养条件下,菌群的群落结构发生了很大改变,表明不同条件下的固碳优势菌属于不同的种属,但通过测序、序列比对及构建系统发育树后发现,在已测序的16个显著条带中,11个是不可培养微生物,即其只能以共生方式存在,混合培养时,固定CO2的效果可能是多种菌共同作用的结果.这意味着单一纯种微生物的固碳效率可能较低,研究与优化固碳微生物菌群的结构和配比将有利于固碳效率的提升.     

碳酸酐酶在中肋骨条藻光合作用中的作用

探讨了在正常空气条件下生长的中肋骨条藻(Skeletonema costatum)的碳酸酐酶(CA)在其光合固碳中的作用.在中肋骨条藻的胞内和胞外均有CA活性,但胞外CA活性很低.CA抑制剂AZ(乙酰唑磺胺)对中肋骨条藻的光合放氧速率没有明显影响,而CA抑制剂EZ(乙氧苯唑胺)对其光合放氧速率有强烈的抑制作用.EZ的抑制作用使细胞最大光合速率、饱和光强和无机碳亲和力下降,无机碳的补偿点和光呼吸提高,使强光下光抑制作用增强.这些结果表明:中肋骨条藻的胞外CA在其光合作用中所起的作用较小,而其胞内CA通过催化胞内碳库中的HCO-3快速转化成CO2,提高胞内CO2的有效供给,从而提高细胞光合固碳能力和对逆境(高O2、强光和低CO2)的适应能力.     

CO2浓度对条浒苔Rubisco酶聚集蛋白核的影响

以生长快速、细胞具多个蛋白核的大型海藻条浒苔作为材料研究CO2浓度对条浒苔Rubisco酶在蛋白核和叶绿体基质之间迁移的影响.应用金标免疫电镜分子定位技术对Rubisco酶集中蛋白核程度进行数值化分析.电镜下可观察到标记Rubisco的金颗粒大部分集中分布在蛋白核中.根据Morita(1997)提出的方法,设定PR-ratio值(蛋白核内分布的Rubisco酶总量与蛋白核外类囊体基质中的Rubisco酶总量之比)作为衡量Rubisco集中蛋白核程度的分析指标.不同CO2浓度对于Kubisco酶分布的长期影响和短期影响研究均显示CO2浓度升高时,Rubisco倾向于向叶绿体基质中扩散;CO2浓度较低或无CO2培养时,Kubisco酶不断向蛋白核中集中.研究结果显示,蛋白核可能在光合作用和CCM机制中具有重要作用.     

光强对两种硅藻光合作用、碳酸酐酶和RubisCO活性的影响

为研究海洋浮游硅藻光合固碳能力与光强的关系, 以三角褐指藻和威氏海链藻为实验材料, 测定了不同光强培养下三角褐指藻和威氏海链藻生长、光合特性、碳酸酐酶和核酮糖-1, 5-二磷酸羧化/氧化酶活性(RubisCO)的变化, 结果显示高光强促进两种硅藻的生长, 但对威氏海链藻的影响更明显。高光强导致两种硅藻叶绿素a、c含量、光系统Ⅱ的最大光化学效率和实际光化学效率明显下降, 非光化学淬灭系数明显升高, 但对光化学淬灭系数并没有明显影响。在高光下威氏海链藻和三角褐指藻胞内外碳酸酐酶活性明显升高。在高光强下培养的威氏海链藻RubisCO活性明显高于低光下培养, 但三角褐指藻正好相反, 不管高光还是低光培养威氏海链藻RubisCO活性始终高于三角褐指藻。以上结果表明不同硅藻对光强变化的响应存在差异, 它们可以通过调节光合生理特征、光合固碳关键酶和CO2供应以适应光强的变化。       

CO2 concentrating mechanisms in cyanobacteria: molecular components,their diversity and evolution

Cyanobacteria have evolved an extremely effective single-cell CO(2) concentrating mechanism (CCM). Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO(2) around Rubisco during photosynthesis. The CCM components include at least four modes of active inorganic carbon uptake, including two bicarbonate transporters and two CO(2) uptake systems associated with the operation of specialized NDH-1 complexes. All these uptake systems serve to accumulate HCO(3)(-) in the cytosol of the cell, which is subsequently used by the Rubisco-containing carboxysome protein micro-compartment within the cell to elevate CO(2) around Rubisco. A specialized carbonic anhydrase is also generally present in this compartment. The recent availability of at least nine cyanobacterial genomes has made it possible to begin to undertake comparative genomics of the CCM in cyanobacteria. Analyses have revealed a number of surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of Rubisco present (Form 1A and 1B). Secondly, the two HCO(3)(-) and CO(2) transport systems are distributed variably, with some cyanobacteria (Prochlorococcus marinus species) appearing to lack CO(2) uptake systems entirely. Finally, there are multiple carbonic anhydrases in many cyanobacteria, but, surprisingly, several cyanobacterial genomes appear to lack any identifiable CA genes. A pathway for the evolution of CCM components is suggested.     

CO2 fixation kinetics of Halothiobacillus neapolitanus mutant carboxysomes lacking carbonic anhydrase suggest the shell acts as a diffusional barrier for CO2

The widely accepted models for the role of carboxysomes in the carbon-concentrating mechanism of autotrophic bacteria predict the carboxysomal carbonic anhydrase to be a crucial component. The enzyme is thought to dehydrate abundant cytosolic bicarbonate and provide ribulose 1.5-bisphosphate carboxylase/oxygenase (RubisCO) sequestered within the carboxysome with sufficiently high concentrations of its substrate, CO(2), to permit its efficient fixation onto ribulose 1,5-bisphosphate. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO(2)-requiring mutant that is devoid of carboxysomal carbonic anhydrase were compared. The kinetic constants for the carbon fixation reaction confirmed the importance of a functional carboxysomal carbonic anhydrase for efficient catalysis by RubisCO. Furthermore, comparisons of the reaction in intact and broken microcompartments and by purified carboxysomal RubisCO implicated the protein shell of the microcompartment as impeding diffusion of CO(2) into and out of the carboxysome interior.     

A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.     

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.     

微藻固定CO2研究进展

空气中CO2浓度升高所导致的温室效应已成为重大的环境问题,受到人们普遍关注.概述了高效固定CO2微藻藻种的筛选和培养方法,分析了微藻固定CO2的无机碳利用形式和浓缩机制,讨论了高效光生物反应器设计和运行目标,简要介绍了微藻(酶)-膜生物反应器集成新技术.并认为今后的研究方向主要是在进一步探索微藻固定CO2有关机理的基础上,构建高效固定CO2的转基因微藻,开发高效膜生物反应集成系统.     

Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.     

The response of Synechococcus PCC7942 (Cyanophyta) to changes in CO2 supply in relation to the acclimation of the CO2-concentrating mechanism. I: physiological study

Distinct types of carboxysomes were distinguished in Synechococcus PCC 7942: electron-clear, electron-intermediate, carboxysomes with internal electron-clear areas, typical electron-dense and bar-shaped carboxysomes. Immunogold location with antibodies against the Rubisco large subunit showed specific label in all carboxysomes. The positive correlation between electron-density, the density of immunogold label, and the percentage of labeled structures within each type support a model of carboxysome biogenesis whereby electron-clear evolve to electron-intermediate and then to electron-dense carboxysomes by the progressive sequestering of Rubisco molecules. Cells responded to limitation in CO₂ supply by increasing carboxysome frequency and the proportion of typical electron-dense carboxysomes, the extent of the response depending on the degree of limitation. The time course of carboxysome expression during transfers between different conditions of CO₂ supply indicated that, under our experimental conditions, there were different levels of response, depending on the degree of limitation. The first level occured at atmospheric levels of CO₂ and involved changes in the affinity of the CCM and in carboxysome, which occurred simultaneously. More severe limitation of CO₂ supply affected carboxysomes exclusively, without further improvement in the affinity of the CCM.