Response Mechanisms To High Carbon During Photosynthetic Reactions Of Microalgae With CO₂ Fixation From Flue Gas

Microalgae have high photosynthetic carbon fixation efficiency,fast growth rate and outstanding economic value.However,there is a serious lack of theoretical understanding of the physiological effects of high concentration of CO₂ molecules on microalgae cells,and a general method with clear theoretical basis and stable experimental results to generally improve the tolerance of microalgae to high concentration of CO₂.Besides,microalgae research is usually in the empirical experimental stage and lacks unified theoretical evaluation methods,resulting in many typical experimental phenomena that are contradictory and difficult to explain in the laboratory and industry.Therefore,in this paper,a variety of experimental methods,such as fluorescence quantitative PCR,flow cytometry and photosynthetic fluorescence measurement,as well as multi-omics combined analysis（such as genomics,transcriptome,proteomics,metabolomics and single-cell sequencing）and bioinformatic data mining methods were adopted.The results revealed that CO₂ gradient acclimation induced multi-target gene mutations to form an adaptive mechanism centered on light response to improve the ability of microalgae to tolerate high CO₂ concentrations,and analyzed the genetic differences of microalgal strains induced by long-term acclimation with different CO₂ concentrations.Three states of carbon deficiency,moderate carbon and excessive carbon were defined according to the light reaction capacity of microalgae cultivation system,and their characteristics of subcategories,photosynthesis,carbon accumulation and metabolism,key enzymes and signal mechanism were revealed.The formula of photosynthetic electron transfer efficiency was modified by measuring the proportional coefficient of microalgae light absorption using f2 colorimetric method.Based on it,the concepts of photoreaction dynamic potential threshold and biological similarity were proposed.The theoretical calculation model for predicting microalgae growth and carbon fixation during photosynthesis was established and a calculation software was written to guide reactor design and actual industrial production.The microalgae Nannochloropsis oceanica CCMP1779 was gradually acclimated to 0.04%,3%,6%,10% and 12% CO₂ to obtain strains with a predominant adaptation to CO₂.It was revealed that the CO₂ gradient domestication produced multi-targeted genetic mutations and natural selection,leading to successful transcriptional changes to enhance photosynthesis in the competition between photosynthesis and respiration.A self-adapted system to high concentration of CO₂ centered on light response was established and supported by improved cation transport,DNA and RNA synthesis,photosynthetic electron transfer,carbon fixation and metabolism.Therefore,CO₂ gradient acclimation was demonstrated as a cost-effective method for generating microalgae mutants for efficient CO₂ fixation from coal-fired flue gas.The effect of long-term high CO₂ stress（6–99%）on the mutation adaptation of microalgae was first clarified with genome and transcriptome analyses.Although longterm high carbon domestication promoted gene mutations in microalgae,at the same time,> 60% CO₂ acclimation caused genotoxicity of microalgae cells via the following mechanisms:（1）it was not conducive to forming more stable long-fragment Indel/SV gene mutations,thus preventing further gene mutation;（2）gene mutations did not generate successful linkage to gene transcription due to inhibition of transcription and protein translation;and（3）inhibition of mismatch repair damaged the specialized ability of genetic variation,leading to unrepair of harmful gene mismatch and less beneficial gene mutations.A theoretical framework was established for explaining the physiological effects of high concentrations of CO₂ on microalgal cells.Three physiological models were established to clarify the photosynthetic features,regulatory systems,metabolic mechanisms and key enzymes of N.oceanica based solely on slightly-high,moderately-high and ultra-high carbon concentrations（6%-99% CO₂）in an univariable environment.Under slightly-high carbon condition,the inhibition of auxin synthesis and an increase in sedatives and hallucinogens due to tryptamine metabolism regulation induced stagnation of biomass accumulation,cell growth and division.However,cell division,carbon and protein metabolism became too active at ultra-high carbon concentrations,which induced multiple cell death factors,including cell integrity destruction from expansins,insulin resistance and hepatotoxin D-galactosamine toxicity.Moderately-high carbon achieved the optimal microalgal biomass yield and fixed carbon content in dried biomass,because the moderate carbon condition slowed down cell division;provided an active carboxylic acid cycle to store abundant CO₂;initiated an effective urea cycle and ammonia transport to inhibit D-galactosamine generation;and induced DNA repair and anti-oxidant flavonoids to eliminate damage from the high CO₂ concentration.The results showed that the optimal CO₂ concentration for the growth and carbon fixation of microalgae varied with light intensity.The ideal combination of CO₂ concentration and light intensity promoted the expression of light-harvesting antenna protein and other proteins in the photosynthetic system,thus improving the photochemical reaction ability and carbon metabolism ability,and finally increasing the microalgal biomass yield.However,mismatched combination of CO₂ concentration and light intensity inhibited carbon fixation and biomass accumulation by slowing down material exchange and nutrient absorption.When CO₂ supply was weaker than light supply,the proportion of premature cells increased;When the CO₂ supply became stronger than the light supply,excess CO₂ intensified the material peroxidation tendency.This research created a novel calculation model for microalgal growth prediction in any cultivation systems.It first proposed a new method to measure and estimate the actual f2 of microalgae in the formula of photosynthetic effective electron transfer rate,ETR=PFD·Φ_Ⅱ·f₁·f₂,by using only a simple spectrophotometer during regular experimentation,replacing complex and expensive instruments.Then based on it,a mathematical relationship between photosynthetic electron transfer and microalgal growth rate was constituted,which even could be simply depicted by a linear formula and concentrated the influence of other cultivation conditions like nutrition and CO₂ into the slope and intercept of this formula.Thus,it could be used to depict any growth conditions.Through manufacturing a C++ software to extend the above relationship to stimulate any 3^-D microalgal cultivation system,a series example cases were made to clarify the significance and utilization of this theoretical model and stimulation method through proving by theoretical calculation and relevant experimental results:（1）Average ETR of the whole cultivation system decided the microalgal tolerance ability to high concentration of CO₂;（2）A photobioreactor located in a certain place（with a certain light condition）had a “theoretical maximum microalgal biomass yield”,which the microalgal biomass could never overcame no matter how the other cultivation conditions was changed;（3）A new concept named “biological similarity” was proposed as the basic principle for magnifying microalgae experiment;（4）a low-cost “dilution method” was proposed to optimize microalgal biomass yield in a short cultivation period.Typical CO₂ carriers of methanol,NaHCO₃ and CO₂ were combined with 15% CO₂ flue gas,then compared for their effects on N.oceanica intracellular metabolic and biological regulation.In the presence of CO₂ gas with and without its ionized acid（HCO₃^-）,microalgal growth was inhibited due to carbon transport-dependent ion transfer and balance.In a weakly acidic environment,growth inhibition was due to cell apoptosis,while in a weakly alkaline environment this was led to cell division inhibition by a decrease of translation initiation factors and carbon fixation stagnation by mass closure of photosynthetic reaction centers.When methanol was combined with 15% CO₂ gas,less negative effects on N.oceanica growth due to changes in the CO₂/HCO₃^-equilibrium.The presence of methanol increased light-dependent reactions by providing sufficient substrates for photosynthetic pigment synthesis through enhanced glycolysis.Total carbon fixation was further increased by higher rates of cell division.The influence of H₂ S impurities in flue gas on cell division,photosynthesis and lipid accumulation of N.oceanica were first investigated.Low concentration of H₂ S effectively promoted synthesis pathways of DNA and RNA,translation initiated by e IF-2 with ribosomal proteins,and cytoplasmic division guided by FtsZ.H₂ S also enhanced the cellular light absorption capacity and electron transfer ability,which promoted cell division and growth under CO₂ fixation.Enzyme analysis showed that low concentration of H₂ S enhanced glycolysis and fatty acids synthesis,as well as suppressed the desaturation,elongation and degradation of fatty acids during intracellular carbon metabolism.Additionally,H₂ S transformed the main ATP synthesis pathway from oxidative phosphorylation to photophosphorylation and substrate phosphorylation,facilitating efficient energy supply during lipid accumulation.Therefore,this markedly increased the yields of fatty acid methyl esters（FAME）and the proportion of short chain FAME.Based on the above research results and theoretical calculation model,in 1200 m² raceway ponds in Yantai Hairong Microalgae Company Co.,LTD.,the goal of producing dry microalgal powder with a 35% proportion of lipid was achieved on an industrial scale without additional cost.Thus,it helped to successfully build a demonstration industrial project of converting CO₂ from flue gas to biodiesel with microalgal photosynthetic biomass accumulation.