Physiological characteristics, hydrogen partial pressure traits and microbial community analysis of a novel low-pH hydrogen producing microbiome

The most widely practiced approach for fermentative hydrogen (H2) producing bio-reactors involves process control of temperatures between 30-41°C, controlled and moderate pH ranges of 4.5-7.5 pH, low headspace and H2-partial pressures. These controls require significant supplemental energy resources that reduce system operating efficiencies. This research discovered a novel multi-species, low-pH H2-producing microbiome, from a heat treated compost inoculum, with optimal operational conditions of: low pH (pH 3.8-3.6 pH) without buffering or pH adjustments, ambient operational temperatures of 22°C to 10°C, higher H2 production under barophilic conditions (1-2 atmospheres above ambient) and greater H2 production at 100% H2 partial pressure (>1 atmosphere) when compare to both 100% N2 partial pressure (>1 atmosphere) and normal intermittent headspace gas release conditions (variable 0 to >2.0 atmospheres). Average bioreactor H2-to-hexose conversion efficiencies were 1.65 mol H2 / mol hexose with a maximum 24-hour rate of 2.95 mol H2 / mol hexose.;The microbial community structure of a low-pH H2-producing microbiome was explored with medium diversity community structure analysis with the bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP ©) process using two 16S rRNA primer sets, 27F-519R (Region 1-3) and 530F-1100R (Region 4-6), in conjunction with an rpoB primer 2700F –3130R. Duplicate samples from a single time point and from one reactor, were analyzed by two 16S rRNA primer sets initially identifying a Clostridium species and Sporolactobacillus species as representative of >98% of the bacterial community (79.46% and 18.57% respectively). However, the rpoB amplicon produced a microbiome bacterial profile of >96% Bacillus species and no other members in common with the two previous 16S rRNA surveys. These results prompted creation of a second set of (Region 2-3) 16S rRNA primers; PF1, a Bacillus biased primer set and PF2, a Clostridium biased primer set, both with a common reverse primer. PF1 detected Clostridium pasteurianum (6.93%), Sporolactobacillus laevolacticus (92.69%), and Bacillus subtilis (0.01%) and PF2 detected Clostridium pasteurianum (99.37%) and Sporolactobacillus laevolacticus (0.14%) but failed to identify Bacillus subtilis as a potential low-pH H2 microbial community member.;Operation of this low-pH H2 producing microbiome provides a net system energy gain over previous systems, and potentially offers a viable H2 production methodology for positive net-energy generation of biohydrogen.