Microbial insights towards understanding the role of hydrochar in alleviating ammonia inhibition during anaerobic digestion

Anaerobic digestion (AD) is known to be inhibited by high concentration of ammonia. The present study proposed and demonstrated the utilization of hydrochar, which is produced by hydrothermal liquefaction of biomass, to alleviate ammonia inhibition. Three different concentrations (0.5, 4 and 8 g/L) of ammonia were tested with acetate as substrate. It was found that hydrochar significantly reduced the lag phase and promoted the methane production rates. The methane production rate was increased by around 10% at both 0.5 and 4 g/L ammonia, while it was increased by as high as 220% at 8 g/L ammonia with the addition of hydrochar. Although hydrochar adsorbed certain amounts of organics and ammonia, the adsorption did not have apparent positive effect on methane production rate. 16S rDNA analysis revealed that hydrochar increased the α-diversity at higher ammonia concentration, and enriched different microbes at each ammonia concentration. Considering the significant increase of methane production rate at 8 g/L ammonia, proteomic analysis was also conducted. Although 16S rDNA analysis showed hydrochar resulted in the enrichment of Clostridium, Methanobacterium and Methanosarcina, slightly different results were obtained by proteomic analysis, which showed hydrochar enriched Clostridium, Methanosarcina and Methanosaeta. The growth of Methanosarcina was inhibited at 8 g/L ammonia, while hydrochar facilitated its growth. The proteins of Methanosarcina involved in both hydrogenotrophic (HM) and aceticlastic methanogenesis (AM) pathways were upregulated by hydrochar. The proteins involved in HM pathway of strictly aceticlastic methanogen Methanosaeta was found to be up-regulated with hydrochar, and it indicated Methanosaeta was involved in direct interspecies electron transfer (DIET), which might be related with the enhanced methane production rate. In addition, Wood–Ljungdahl (WL) pathway of Clostridium was also found to be upregulated with hydrochar. Clostridium might be the bacteria that provide H+ and e- to Methanosaeta for DIET considering its enrichment with hydrochar.