Effects of palm oil mill effluent anaerobic sludge pretreatment temperature on biohydrogen production and the inoculum microbial community

Abstract

Biohydrogen production yield from dark fermentation could be improved by inhibiting interspecies hydrogen transfer to methanogens, homoacetogens and suppress non-biohydrogen producers as metabolic competitors. Heat pre-treatment has been extensively used for this purpose. In this study, the effects of heat pre-treatment temperatures on the performance of mesophilic biohydrogen dark fermentation system and the inoculum microbial community were evaluated, using POME anaerobic sludge as inoculum. Heat pre-treatment of the anaerobic sludges were conducted at 50°C, 65°C, 80°C and 100°C for 30 minutes. Biohydrogen production was evaluated under batch fermentation (30°C, initial pH 5.5-6.0, 180 rpm). Shotgun metagenomics analysis was carried out on the raw, untreated and treated sludge inoculum after fermentation. The results showed that pre-treatment of the inoculum at 65°C produced the highest biohydrogen yield of 0.67 mol H2/mol hexose followed by pre-treatment at 80°C which produced 0.62 mol H2/mol hexose. Untreated inoculum yielded 0.30 mol H2/mol hexose while 50°C and 100°C pre-treated inoculum produced less than untreated anaerobic sludge. Methane was not detected in any of the fermentation reactions. Shotgun metagenomics revealed that inoculum heat pre-treatment temperatures influenced the microbial communities in biohydrogen production fermentation. Inoculum pre-treated at 65°C and 80°C were enriched with the most biohydrogen-producing taxa, mainly spore-forming Clostridia which generate biohydrogen via butyrate-type fermentation pathways, producing butyric and acetic acids. Untreated anaerobic sludge fermentation was not significantly enriched with biohydrogen-producing microbial taxa. Heat pre-treated inoculum at a lower temperature of 50°C was enriched with non-spore-forming biohydrogen producers from Klebsiella, Escherichia and Citrobacter genera. These genera produced low biohydrogen yield by shifting the fermentation pathway to mixed-acid fermentation, producing a mixture of acetic and butyric acids and ethanol. The presence of non-biohydrogen producers, such as lactic acid bacteria also decreased biohydrogen production of the 50°C pre-treated inoculum, due to the lactic acid and ethanol fermentations. Heat pre-treatment at higher temperature of 100°C selectively enriched spore-forming microbial taxa. Among the species are biohydrogen producers B. coagulans, homoacetogens C. magnum, and non-biohydrogen producers from Bacillus species. These species re-direct the fermentation pathway to mixed-acid fermentation and produced high concentration of acetic acids. Methanogens, e.g. M. soehngenii present in the raw anaerobic sludge were suppressed in all the fermentation reactions. Metabolic functions analyses from the metagenome data showed that biohydrogen production potentially upregulate functions related to cellular processes (prokaryotic cell spore formation), carbohydrate metabolisms (sugar alcohols, monosaccharides, sugar acids and carboxylic acids metabolisms) and energy (fermentation and methanogenesis). In conclusion, inoculum heat pre-treatment is essential to enhance biohydrogen production using POME anaerobic sludge as heat pre-treatment enriched the inoculum with biohydrogen producers and suppress activities of methanogens, non-biohydrogen producers and homoacetogens.