| Lactic acid, as a naturally occurred organic acid, is widely applied in food, chemical and pharmaceutical industries. Recently, with the improvement of environmental awareness, products from polylactic acid become substitute for traditional plastics, and it greatly promotes the global lactic acid market. However, many key problems still remained to be solved during microaerobic lactic acid fermentation process, such as difficult to characterize and regulate oxygen metabolism, low fermentation efficiency as well as high byproduct levels. In this paper, based on basic physiological characteristics of potential industrial strain (Lactobacillus paracasei), and integrated with multi-scale parameters association analysis methodology, intra-and extracellular physiological and chemical responses, key enzyme acitivities determination, intracellular metabolite profiling and metabolic flux analysis as well as the integration of broth rheological properties were investigated to reveal possible oxygen metabolism mechanism and cellular osmotic stress responses in microaerobic lactic acid fermentation by L. paracasei. Consequently, rational process regulation in lactic acid fermentation was achieved to improve titer, productivity and yield under high initial glucose condition.Firstly, basic physiological characteristics of L. paracasei was investigated. The results demonstrated that homolactic fermentation occurred under microaerobic conditions. Lactic acid formation was closely related to cell growth with a correlation coefficient of 6.23, and an uncorrelation coefficient of 0.18. During the fermentation, neutralizing agent addition amount, which had good linear relation to lactic acid titer, could be utilized to on-line monitor key parameters during the process. Moreover, physiological parameter, carbon dioxide evolution rate (CER) could be used to feature cell growth. With the guidance of CER and respiratory quotient (RQ), strategy for nitrogen source step-wise addition was adopted with enhancements on productivity and yield by 5.94% and 3.05%, respectively, while byproducts acetoin and acetate reduced by 24.68% and 31.25%.Secondly, systemically investigation on the mechanism of cell oxygen metabolism was conducted and following two-step OUR control strategy was adopted for process optimization. Due to the microaerobic fermentation process, conventional oxygen metabolism parameters, dissolved oxygen (DO) and oxidation-reduction potential (ORP). showed their obvious limitations. In the broth. DO was always lower than the detection limit, while ORP was affected by other oxidative and reductive materials except for oxygen. Here, we introduced physiological parameter, oxygen uptake rate (OUR) to characterize oxygen metabolism for the first time with the application of exhaust gas mass spectrum. Simultaneously, on the basis of oxygen metabolism during the process, OUR levels could be quantitatively regulated through aeration. Subsequently, the effects of OUR on cell growth and metabolism were studied. The detailed results were summarized as follows:(1) oxygen was consumed by both NADH oxidase and electron transfer chain (ETC). The former mainly functioned at early growth phase, while the latter played its role at late growth and stationary phases. (2) with the precondition of homolactic acid fermentation (yield more than 80%), high OUR levels during the growth phase could promote cell growth, glucose consumption as well as lactic acid production, while it decreased yield. (3) during growth phase, metabolic flux from pyruvate to acetyl CoA enhanced significantly with the increase of OUR level, which would reversely result in lactic acid yield decrease. Simultaneously, compared to PFL, PDH also exhibited its important role on metabolizing pyruvate to acetyl CoA with high OUR level. (4) high OUR could make NAD+ regeneration more flexible and enhance acetate formation (ATP-yielding pathway), which then advanced glucose metabolism and energy synthesis for cell growth. (5) OUR increase mainly played role on pyruvate metabolism, shifting from lactic acid to acetyl CoA node. Furthermore, cofactor balance was achieved mainly through regulating flux ratio of acetate and ethanol. (6) when cell entered into stationary phase, intracellular metabolites profiling and key enzyme activities analysis revealed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the control node of glycolysis flux. And high OUR would intensify the accumulation of ROS level, which inhibited GAPDH activity significantly, resulting in the great decrease of glucose metabolism. (7) both decrease of glucose metabolism and lactic acid yield contributed to productivity reduction, moreover, regulation of pyruvate metabolism to lactic acid was occurred on metabolic level, not on enzymatic level, as NADH pool and NAD+/NADH differed significantly, while lactic acid dehydrogenase activities changed little with different OUR levels. (8) intracellular metabolic flux indicated that, similar to cell growth phase, OUR also functioned on pyruvate metabolism. However, during cell stationary phase, cofactor balance was principally dependent on acetoin node, not acetyl CoA node. (9) profile of NAHD utilization illustrated that NADH used for lactic acid synthesis decreased (from 94.5% to 84.5%), while for byproducts synthesis and ETC consumption enhanced with OUR increased from 0.14 to 0.85 mmol/L/h. (10) through ATP synthesis efficiency and its composition analysis, it could be found that cell would produce more energy through byproduct pathway and ETC to response stronger oxidative stress under high OUR condition. Based on above-mentioned results, we established two-step OUR control strategy to improve lactic acid productivy by 12.7%, compared to keeping OUR level at 0.14 mmol/L/h. and enhance lactic acid yield by 4.04%. compared to keeping constant OUR level at 0.43 mmol/L/h. Furthermore. bubble size distribution, oxygen transfer coefficient (KLa) and oxygen transfer rate (OTR) were greatly dependent on broth rheological characteristic, which ascribed to the higher OUR level and better fermentation performance with calcium hydroxide as neutralizing agent, compared to ammonium hydroxide and sodium hydroxide.Subsequently, effects of osmotic pressure and neutralizing agent on lactic acid production were studied. Under high initial glucose conditions, neutralizing agent not only presented effect on OTR and OUR levels, but also functioned on environmental osmotic pressure. Low calcium lactate solubility and severe osmotic stress caused by ammonium lactate were responsible for significant productivity decrease at later phase of fermentation, not accumulation of NH4+ or LA" as well as lactic acid molecule. Investigation by shake flask and verification by 5 L bioreactor demonstrated three pivotal osmotic levels would affect cell growth and metabolism by L. paracasei. Below 3000 mOsm/kg, cell could metabolize normally, but growth would be slightly and significantly inhibited when osmotic pressure reached 1800 and 3000 mOsm/kg, as long as it exceeded 3600 mOsm/kg, both growth and metabolism almost ceased. Through sequential analysis of membrane fatty acid composition, it was found that with osmotic pressure increase, ratio of unsaturated and saturated fatty acid raised greatly, same for epoxyfatty acid proportion. Additionally, intracellular metabolite profiles indicated that intermediate pools in EMP, TCA and PPP pathway decreased significantly, while some potential osmotic protectants (glycerol, mannitol, proline and aspartate) showed high enhancement. Consequently, exogenous proline could be taken to accumulate in the cell, and 2 g/L proline was proved to have the best effect on resisting osmotic stress within certain range (3000-3600 mOssm/kg), while as far as osmotic pressure exceeded 3600 mOsm/kg, its protective role became poor. Other than cell tolerance improvement, process control strategy also could alleviate environmental osmotic stress. Here, neutralizing agent combination was developed, which could not only avoid calcium lactate crystallization, but also decrease osmotic pressure efficiently, with an improvement on productivity by 2.21-fold and a slight enhancement on yield, compared to ammonium hydroxide as neutralizing agent.Lastly, based on a newly developed probe, we explored NADH changes in L. paracasei. Although NADH plays an important role on lactic acid production, its real-time, dynamic and high spatio-temporal resolution determination still perplexed the researchers. Here, a genetically encoded fluorescent probe (Frex), which developed by Yang’s group, was introduced into lactic acid bacteria for preliminary application. It was found that recombinant strain could effectively represent physiological characteristics of the parent strain, although it showed weaker metabolic capacity. Through investigation of its responses to various environmental stresses, we approved that Frex was specific to NADH determination, and it is feasible to be used in L. paracasei. Adoption of Frex for real lactic acid fermentation processes with different OUR levels verified the important role of oxygen metabolism on regulating lactic acid fermentation again. Hopefully, Frex, as a powerful in vivo NADH detection tool, could be widely applied in other bioprocesses. |
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