Solid-phase denitrification is one of the most promising methods for improving nitrate removal that relies on heterotrophic denitrifiers, and the availability of organic carbon significantly influences the denitrification efficiency. Synthetic carbon sources are often costly; on the other hand, natural carbon substrates are known for their abundant bioavailability. Selecting the right carbon source is crucial for achieving optimal denitrification efficiency. However, the underlying research on elucidating the efficiency of natural substrates for denitrification are limited. Therefore, the present study evaluated the denitrification potential of agricultural wastes, including rice straw (RS), wheat straw (WS), tender coconut shells (CS), and a combination of these thereof under batch and continuous mode. The study revealed that the continuous flow mode yielded the highest denitrification efficiency of 96.13%. This result outperformed the individual carbon sources' efficiencies in batch mode, which were 72.6% (WS), 60.3% (CS), and 58.6% (RS). The superior efficiency of the continuous mode was attributed to factors such as increased biomass and stable biofilm formation. Physiochemical changes of lignocellulosic biomass in terms of distorted surface morphology following microbe-based cellulose hydrolysis were observed through scanning electron microscopy (SEM). Increase in elemental carbon and decrease in oxygen content, as evident from energy dispersive X-ray spectroscopy (EDS), further supported biofilm formation following denitrification. Changes in cellulose structure were also observed through changes in surface functional groups and crystallinity via fourier transform infrared spectroscopy (FTIR) and X-ray diffractogram (XRD) respectively. Initial visualization and microscopic identification of microbes from the denitrifying column showed the bacterial strains to form opaque, red, and yellow colonies with characteristic rod-shaped and gram-negative nature. Phylogenetic analysis through 16S rRNA sequencing identified the strains as Rhodococcus ruber and Cellulosimicrobium cellulans. Overall, the findings provide insights into the effective utilization of agricultural waste as a solid carbon source for enhancing denitrification efficiency in wastewater treatment processes.
