Nitrogen-deficient cultivation enables energy-efficient bio-flocculation of purple non-sulfur bacteria at native pH

Abstract: Purple non-sulfur bacteria (PNSB) are promising feedstocks for wastewater treatment and bio-refinery applications. However, inefficient harvesting is a major barrier to realizing their large-scale applications. This study demonstrates that nitrogen-deficient (ND) cultivation is an effective strategy for enhancing PNSB bio-flocculation, compared with traditional nitrogen-sufficient (NS) cultivation. For both NS and ND cultures, chitosan-induced flocculation was evaluated across a range of pH values (5.0-8.0) and polymer dosages (20-300 mg/L). Under NS conditions, flocculation efficiency (F.E.) reached 71-75% under acidic pH (5.0-6.0) and declined to <50% at pH 7.0-8.0. In contrast, ND culture achieved 83-86 % F.E at pH 7.0-8.0. It corresponds to the native pH (~8.0) of both ND and NS growth media, thereby eliminating the need for chemical addition to adjust the pH. At this pH, the maximum F.E. (72%) was achieved at 100 mg/L of chitosan, whereas NS cultures required a higher dosage (200 mg/L) to reach a lower maximum F.E. (61%). In ND cultures, the zeta potential shifted from -30 to -12 mV, indicating that polymer bridging was the dominant flocculation mechanism. ND culture also showed significantly higher baseline hydrophobicity (40% vs 17% in NS), which increased to 90% at 250 mg/L of chitosan. It also found that the biomass concentration strongly influenced flocculation, with undiluted ND culture showing higher F.E. (80%), and it reduced to only 15% at 5x dilution. FTIR spectroscopy, microscopic analysis, and microbial community analysis confirmed that enhanced flocculation in ND culture was attributed to an increase in extracellular polymer substances. Thus, ND cultivation enables higher F.E. at native and near-neutral pH levels and at lower polymer dosages, demonstrating its potential to enhance the techno-economic viability of PNSB-based biorefineries