Rubisco’s low affinity for external CO₂ drove the evolution of CCMs. Therefore, one of the most useful indicators of whether a photosynthetic organism has a CCM is to compare the affinity of photosynthesis for external CO₂ with the affinity of Rubisco for CO₂. This approach was originally applied to higher plants, and by measuring the affinity K_m (Rubisco CO₂) of Rubisco extracted from photosynthetic organisms to substrate CO₂, it was inferred that C₃ species might require a CCM [69]. However, due to the limitation of extraction technology, the affinity determination of the photosynthetic organism Rubisco lacked accuracy. With the improvement of extraction and analysis techniques, this method has become widely used to evaluate algal CCMs. In general, the researchers measured the photosynthetic oxygen evolution rate under different inorganic carbon concentrations and used the data analysis software to fit the inorganic carbon response curve (P-C curve) by using Michaelis–Menten equation, where K_m is the substrate concentration (DIC concentration) when the photosynthetic rate is half of the maximum, from which we can infer the affinity of algae photosynthesis to CO₂ K_m (photosynthetic CO₂). Since marine algae may experience more persistent CO₂ limitations than fresh- and brackish-water autotrophs, it can be assumed that the type of water environment determines the effectiveness of CCM. Comparing the in vivo photosynthesis and the in vitro carboxylation of Rubisco under ambient oxygen levels can show its ability to concentrate CO₂ exceeds the level of environmental CO₂ [70]. For example, C. reinhardtii’s ratio of K_m (photosynthetic CO₂) to K_m (Rubisco CO₂) is approximately 1:30, and that of Ulva sp. is about 5–10:68 [1,71,72]. Both apparently rely on CCM to improve the efficiency of Rubisco, whereas other algae with close specific gravity may not use CCM.

Because CA is central in all chloroplast CCM models, determining the effect of CA inhibitors is useful in studying the functional aspects of algal CCM. This is especially true for chloroplast-penetrating inhibitors, such as EZA (Ethoxzolamide), which are likely to inhibit most forms of internal CA. EZA is often used to obtain low inorganic carbon affinity for photosynthesis of algae. If the algae showed low inorganic carbon affinity under the action of EZA, it is proved that there is intracellular CA-mediated CCM in the algae, but no effect may not be conclusive evidence of CCM deletion. For CA inhibitors that cannot penetrate cell membranes, such as AZA (Acetazolamide), only apply to inhibit the activity of external CA [47], the effect on the function of chloroplast CCM is less easily explained. By comparing the relative effects of EZA and AZA, the relative contributions of internal and external CA forms to photosynthetic Ci absorption can be determined (Figure 3). For example, in the study of Gao et al. [73], after adding AZA the net photosynthetic rate of U. linza decreased, and the inhibition rate was noted to be 26.26%. Compared with AZA, EZA demonstrated a higher inhibition rate of photosynthesis (75.19%), which indicated that the internal CA contributed more to the absorption of photosynthesis. Xu et al. [74] found that U. linza and U. prolifera have obvious extracellular and intracellular CA activity by using different CA inhibitors. However, extracellular CA enzyme activity itself accelerates the mutual conversion between HCO₃⁻ and CO₂ but cannot affect the CO₂ equilibrium concentration, and CO₂ mainly enters the cell through passive diffusion. This indicates that HCO₃⁻-utilization in this manner does not function well at high pH (pH > 9.4) because the equilibrium concentration of CO₂ is low. It is obvious that in addition to the extracellular CA catalytic utilization of HCO₃⁻, there must be another means of using HCO₃⁻ in U. prolifera.

HCO的功能₃⁻在光合作用中被认为是藻类的一种特性[75]。HCO的利用₃⁻藻类取决于外部CA和质膜转运蛋白的参与[76,77,78]。HCO的使用₃⁻与 CCM 的存在密切相关。然而，一些大型藻类不仅能够利用HCO₃⁻作为碳源，一些人已经证明了一氧化碳的使用₂在特定情况下。通过检测δ¹³C同位素在石莼和石莼中，发现当环境CO浓度₂很高，使用HCO有生理转变₃⁻几乎完全主要使用一氧化碳₂.在当前的海水中 pCO₂，许多大型藻类使用HCO₃⁻而不是溶解的一氧化碳₂并利用CA转换HCO₃⁻到 CO₂供鲁比斯科使用[79,80,81,82]。例如，Mercado等人[83]发现，在当前的海水CO下₂水平，叶绿素植物刚性狸藻和压缩狸藻不能获得足够的CO ₂仅通过扩散吸收;因此，必须使用CCM来获得HCO₃⁻.然而，大型藻类可能会下调其CCM，降低HCO₃⁻使用，并变得依赖一氧化碳₂作为CO时的主要碳源₂浓度高[1,84,85,86]。因此，当使用这些指标评估石莼属的CCM时，HCO的使用可能会减少₃⁻在某些情况下，仍然存在CCM活动，但有一定程度的下调。

当外部环境中存在Ci限制时，Ci转运蛋白对外部CO的亲和力₂和 HCO₃⁻增加，细胞内和细胞外CA活性也增加。两者共同作用，使微藻对外部Ci的亲和力增加了10倍以上[76]。这种可诱导的变化似乎是蜂窝基础设施参与供应一氧化碳的有力证据。₂到叶绿体中的鲁比斯科。这些诱导CCM不仅限于微藻，而且在许多大型藻类中也观察到，包括石莼[1]，格拉西拉里亚[87]，卟啉[88]和墨角[89]。也就是说，一些藻类在面对外部Ci周期约束时可能具有诱导CCM来满足其环境需求，而其他藻类可能没有这种灵活的排列。