Highly efficient biohydrogen production by diverse microbial community in landfill leachate sludge using dark fermentative batch reactor

This study investigated the microbiology in landfill leachate sludge for biological H2 production. This sludge originated from sanitary landfill, hence the microbial community which survived in the sludge may have unique H2-producing features. The landfill leachate sludge was pretreated at different temperatures and landfill leachate sludge pretreated at 65 ºC revealed the maximum H2 yield of 6.43 ± 0.16 mol H2/mol glucose under the optimum conditions of 37ºC and pH 6. This new record of high H2 has exceeded the conventional theoretical yield of 4 mol H2/ mol glucose. Under the same optimum condition (37ºC and pH 6), the H2 produced from dairy wastewater was 113.2 ± 2.9 mmol H2/g COD (12.8 ± 0.3 mmol H2/g carb.). The H2 productions from glucose and wastewater were thermodynamically favourable with the Gibb’s free energy of –34 and –40 kJ/mol, respectively. The microbial community was successfully revealed by 16S-rRNA Metagenomics using Illumin Miseq. Sludge pretreated at 65 ºC was revealed to contain 98% of H2-producing bacteria which mainly belong to the genera Clostridium, Bacillus, Eubacterium and Sporacetigenium. In comparison, untreated sludge which contained mainly H2-consuming bacteria including genera Pseudomonas, Sulfurimonas and Treponema. The sludge pretreatment has successfully eliminated H2-consuming bacteria and enriched H2-producing bacteria which in turn improved H2 yield up to 53%. Three H2-producing bacteria were successfully isolated and identified as Clostridium perfringens strain JJC (AWRZ00000000), C. bifermentans strain WYM (AVSU00000000) and Clostridium sp. strain Ade.TY (AVSV00000000). Based on multiple genome alignment, Clostridium sp. strain Ade.TY is likely to be a new H2-producing species as it does not aligned to the genome of its closest neighbour. The batch mode optimization showed that the maximum H2 production attained from these isolates are in the descending order of C. perfringens strain JJC > C. bifermentans strain WYM > Clostridium sp. strain Ade.TY with yield of 4.68 ± 0.12, 3.29 ± 0.11, 2.87 ± 0.10 mol H2/ mol glucose, respectively. This shows that C. perfringens strain JJC could convert substrates to H2 more efficiently as compared to the others. Nonetheless, it is important to note that the H2 yield from single H2-producing bacterium was less efficient as compared to the performance of H2 production using landfill leachate sludge as inoculum. In summary, landfill leachate sludge contains functional microbial community for efficient H2 production with good potential in industrial application.