Anthropogenetic changes and energy generation from fossil fuels having devastating effect on environment. Renewable fuel generation from lignocellulos biomass can contribute in two directions to minimize the GHG emission and sequestration of carbon dioxide.  In first step enzymatic hydrolysis of corn stover was carried out for reforming of biomass to generate biohydrogen. After filtration soluble sugars and enzymatic solution was used to enrich PNSB for hydrogen production under different LED light wavelength to assess the hydrogen and carotenoids assessment. Highest cumulative hydrogen volume (CHV) and the maximum hydrogen production rate (HPR) of 568.8 mL and 9.17 mL/h, respectively were achieved in both reactions. The sampling and extraction procedure was performed as follows: 5–20 mL bacterial fermentation broth was centrifuged at 4,000 rpm (1,800 ×g) for 15 min, then the supernatant was discarded. 1 mL methanol and 2 mL hexane were then added to the pellet, and the mixture was vortexed for 2 min. The method was employed for examining the carotenoid composition observed during growth of PNSB, determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis in PNSB. The relationship between different wavelength of LED and hydrogen production, biomass conversion ratio, and by-products has been found it. Results showed that suitable wavelength not only enhances the biohydrogen production but also effect the overall biomass conversion ratio.