Impact of phosphate limitation on PHBV production and microbial community dynamics fed with synthetic fermentation digestate [ITS]

This study investigates the role of carbon-to-phosphorus (C/P) ratios in shaping microbial community dynamics and polyhydroxyalkanoates (PHA) production in sequencing batch reactors (SBR) fed with volatile fatty acids. Three conditions, characterized by fixed organic loading rates but varying C/P ratios (Run 1 = 170 Cmol Pmol-1; Run 2 = 235 Cmol Pmol-1; Run 3 = 400 Cmol Pmol-1), were tested to explore their impact on PHA accumulation, biomass growth, and reactor stability. Results indicate that the moderate phosphorus limitation of Run 2 achieves the best overall performance, with a PHA volumetric productivity of 2.02 g PHA L-1 d-1 and process stability. Conversely, higher C/P ratio increased the storage yield but lowered the productivity (1.55 gPHA L-1 d-1), compromising sludge settleability and reactor stability, indicating impaired microbial functionality. Full-length 16S/18S rRNA gene sequencing using PacBio technology enabled high-resolution profiling of microbial communities, revealing ecological shifts across conditions. Run 2 exhibited the highest bacterial and eukaryotic diversity, featuring multiple PHA-accumulating bacteria (mainly Sphaerotilus, Leadbetterella, and uncultured Rhodobacteraceae) and a well-structured eukaryotic community dominated by K-strategist bacterivorous protists, e.g. Rhogostoma (Rhizaria) and Vorticella (Ciliophora, Oligohymenophorea) and predatory protozoa, e.g. Tokophrya (Ciliophora, Suctoria). In contrast, Sphaerotilus dominated under suboptimal conditions and may be linked to bulking. These results suggest that eukaryotic diversity may support system stability through selective predation. Transmission electron microscopy (TEM) further confirmed the presence of intracellular PHA granules and polyphosphate reserves, reinforcing the connection between nutrient limitation and adaptive microbial strategies. Overall, these findings highlight the critical role of the C/P ratio in shaping the performance of mixed microbial cultures, demonstrating that a well-balanced nutrient supply can enhance PHA production while maintaining microbial community stability. The results contribute to optimizing the selection process for mixed microbial cultures, offering valuable insights into the impact of carbon-to-nutrient ratios in the feeding strategy. Overall design: Total DNA was extracted from activated sludge and mixed microbial cultures grown under three C/P conditions, then amplified for full-length ITS amplicon sequencing using the PacBio platform

本研究探究了碳磷比（carbon-to-phosphorus, C/P）在以挥发性脂肪酸（volatile fatty acids）为底物的序批式活性污泥反应器（sequencing batch reactors, SBR）中，对微生物群落动态及聚羟基脂肪酸酯（polyhydroxyalkanoates, PHA）合成的调控作用。本研究设置了三种有机负荷率固定但碳磷比各异的运行条件（运行1：170 Cmol·Pmol⁻¹；运行2：235 Cmol·Pmol⁻¹；运行3：400 Cmol·Pmol⁻¹），以考察其对PHA积累、微生物生长及反应器稳定性的影响。结果表明，运行2的中度磷限制条件下整体性能最优，其PHA体积产率达2.02 g PHA·L⁻¹·d⁻¹，且系统运行稳定。反之，较高的碳磷比虽提升了底物储存产率，却降低了PHA产率（仅1.55 g PHA·L⁻¹·d⁻¹），同时恶化了污泥沉降性能与反应器稳定性，提示微生物功能受到损害。采用PacBio技术完成的全长16S/18S核糖体RNA基因测序，实现了微生物群落的高分辨率谱分析，揭示了不同条件下的群落生态演替。运行2的细菌与真核生物多样性最高，其核心菌群包含多种PHA合成菌（主要为球衣菌属*Sphaerotilus*、Leadbetterella属及未培养的红杆菌科Rhodobacteraceae成员），且真核群落结构完善，以K策略食细菌原生生物为主，例如根肿虫属*Rhogostoma*（SAR超类群根瘤菌门Rhizaria）、钟虫属*Vorticella*（纤毛虫门Ciliophora，寡膜纲Oligohymenophorea），以及捕食性原生生物如*Tokophrya*（纤毛虫门Ciliophora，吸管纲Suctoria）。与之相反，在非最优条件下球衣菌属占据优势，这可能与污泥膨胀相关。上述结果提示，真核生物多样性可通过选择性捕食作用维持系统稳定性。透射电子显微镜（transmission electron microscopy, TEM）进一步证实了细胞内PHA颗粒与多磷酸盐储备的存在，佐证了营养限制与微生物适应性策略之间的关联。综上，本研究结果凸显了碳磷比在调控混合微生物培养系统性能中的关键作用，证实适度均衡的营养供给可在维持微生物群落稳定的同时提升PHA合成效率。该研究结果可为混合微生物培养的筛选优化提供参考，为底物投加策略中碳营养比的影响机制提供了宝贵见解。总体实验设计：从三种碳磷比条件下培养的活性污泥与混合微生物菌群中提取总DNA，经扩增后采用PacBio平台完成全长ITS扩增子测序。