Chlorella vulgaris MSU-AGM 14 a freshwater green microalga exhibited photobiological hydrogen production by employing aqueous extract of green seaweed Valoniopsis pachynema. The microalga was isolated from the pond water followed by its screening to study the biohydrogen production capability at anaerobic and sulfur depletion conditions with, 15 mu mol photons m(-2) s(-1) light illumination. The aqueous extract (10-100%) of green seaweed V. pachynema served as carbon and nitrogen source for their active growth and biohydrogen production at varied concentrations. The optimizations of four individual variables (substrate concentration, temperature, pH and carbon dioxide) were examined by using Response Surface Methodology (RSM). The collected biohydrogen gas samples were analyzed quantitatively and qualitatively by using Gas Chromatography (GC). The 16S rRNA proved that the C. vulgaris MSU-AGM 14 has 609 bp fragment within the chloroplast genome of Chlorella Beijerinck genus. The optimized individual variables were obtained at a concentration of 22.5% of seaweed aqueous extract at a medium pH of 6.8, it a temperature of 32 degrees C with 5% carbon dioxide for active photobiological hydrogen production. Collectively, the results demonstrate that biohydrogen production in C vulgaris MSU-AGM 14 were increased by employing aqueous extract of green seaweed Valoniopsis pachynema. (C) 2016 Elsevier Ltd. All rights reserved.