The development and microbiology of bioprocesses for the production of hydrogen and ethanol by dark fermentation

Abstract

This work investigated the production of hydrogen and ethanol from carbohydrates by bacterial dark fermentation. Meso and thermophilic fermenters were enriched from the environment, and their H2 and/or ethanol production in batch determined. Continuous biofilm, suspended-cell and granular-cell processes for H2 or ethanol+H2 production from glucose were developed and studied. Dynamics of microbial communities in processes were determined based on the 16S rRNA gene sequence analyses.

Mesophilic enrichment, obtained from anaerobic digester sludge, produced 1.24 mol-H2 mol-glucose-1 in batch assays. Hydrogen production by the enrichment in a mesophilic fluidized-bed bioreactor (FBR) was found to be unstable  prompt onset of H2 production along with butyrate-acetate was followed by rapid decrease and cease associated with propionate-acetate production. Intermittent batch (semi-continuous) operation allowed a momentary recovery of H2 production in the FBR. The highest H2 production rate (HPR) observed in FBR was 28.8 mmol h-1 L-1, which corresponded to a relatively high hydrogen yield (HY) of 1.90 mol-H2 mol-glucose-1.

Mesophilic, completely-mixed column reactor (CMCR), with a similar inoculum and feed as used in the FBR, provided a prolonged H2 production for 5 months. Highest HPR observed in the CMCR was 18.8 mmol h-1 L-1 (HY of 1.70 mol-H2 mol-glucose-1), while it in general remained between 1 and 6 mmol h-1 L-1. Hydrogen production in the CMCR was decreased by shifts in microbial community metabolism from initial butyrate-acetate metabolism, first to ethanol-acetate, followed by acetate-dominated metabolism, and finally to propionate-acetate metabolism, which ceased H2 production. The transitions of dominant metabolisms were successfully detected and visualized by self-organizing maps (SOMs). Developed Clustering hybrid regression (CHR) model, performed well in modeling the HPR based on the data on process parameters (pH, HRT) and metabolites (organic acids, ethanol, CO2).

The instability of mesophilic processes (FBR and CMCR) was found to be due to rapid changes in microbial community structures after the start-up of continuous operation. The enrichment of organisms in bioreactors changed community metabolism away from H2 (and butyrate-acetate) production. The FBR supported the growth of more diverse microbial community than that observed in the CMCR. Clostridium butyricum was the main H2-producing organism in mesophilic bioreactors based on the metabolic pattern (e.g., high B/A ratio) and on the 16S rRNA gene sequence analyses. The changes in quantities of C. butyricum (based on quantitative real time-PCR, and on proportion trends by DGGE) roughly corresponded to those in HPR.

Hydrogen production was more stable in the mesophilic CMCR than in the FBR. The instability of H2 production in mesophilic reactors was likely related to the following reasons: Improper biocarrier in the FBR (low mass transfer of H2, good adhesion of propionate-producers); unsuitable microbial community, e.g., presence of propionate-producers (BESA enrichment, no selection of spore-formers); too low bioreactor loading (caused sporulation of C. butyricum and favored the growth of propionate-producers).

Thermophilic isolate AK15, affiliated with C. uzonii (98.8%), produced relatively high amounts of H2 from glucose (up to 1.9 mol-H2 mol-glucose-1) and xylose (up to 1.1 mol-H2 mol-xylose-1) in batch at 60C. Batch ethanol production by another thermophilic strain, AK17, affiliated with Thermoanaerobacterium aciditolerans (99.2%), was amongst the highest reported for thermoanaerobes with ethanol yields of up to 1.6 mol-EtOH mol-glucose-1 and 1.1 mol-EtOH mol-xylose-1 in batch assays at 60C. The HYs in batch by AK17 were up to 1.2 mol-H2 mol-glucose-1 and 1.0 mol-H2 mol-xylose-1. Further, AK17 tolerated up to 4%, v/v of exogenously added ethanol, and utilized main sugar residues found in lignocellulosic materials. Stable, long-term (3 months), co-production of ethanol and H2 was achieved in an open system, CMCR by a co-culture of AK15 and AK17 at 60 C. AK17 became dominant in the CMCR, producing promising ethanol yield of 1.35 mol-EtOH mol-glucose-1 and HPR of 6.1 mmol h-1 L-1 from glucose at the HRT of 3.1 h.

Extensive screening of Icelandic hot spring samples with glucose resulted in several H2 and ethanol+H2 -producing enrichment cultures, over a temperature range from 50 to 78 °C. One enrichment produced H2 directly from cellulose at 70 C. Enrichment 9HG, dominated by bacteria closely affiliated with Thermoanaerobacter thermohydrosulfuricus (100%), produced relatively high yields of ethanol (1.21 mol-EtOH mol-glucose-1), and some H2 (0.68 mol-H2 mol-glucose-1), from glucose in batch at 78 °C. Lactate production decreased the ethanol (0.69 mol-EtOH mol-glucose-1) and H2 (0.32 mol-H2 mol-glucose-1) yields in the continuous-flow bioreactor at 74 C, and the yields were lower than those obtained in the batch fermentations. Co-production of ethanol+H2 by 9HG was pH-dependent, and favored at the pH range of 6.5 to 7.1.

The hydrogen yield in batch (3.2 mol-H2 mol-glucose-1) by hot spring enrichment 33HL was among the highest reported for thermoanaerobes. The batch 33HL produced H2 along with acetate. The dominant bacteria in the batch 33HL, Thermobrachium celere (100%) affiliated strains, did not thrive in continuous or semi-continuous open reactor systems fed with glucose. Continuous or semi-continuous reactor cultures with 33HL were dominated bacteria closely affiliated with Thermoanaerobacterium aotearoense (98.5 99.6%). These cultures produced H2 along with acetate and butyrate.

High HY of 2.51 mol-H2 mol-glucose-1 by 33HL was obtained in semi-continuous reactor at the HRT of 24 h at 58C. High hydrogen production rate from glucose, 45.8 mmol h-1 L-1, was obtained in continuous-flow reactor by 33HL at the HRT of 3h. Hydrogen production by 33HL was characterized by higher H2 production efficiency (i.e., higher H2 yield or specific H2 production rate) than reported for mesophilic cultures. The 33HL readily formed granules in the continuous and semi-continuous reactor systems. Possessing good self-granulation, wide substrate utilization range and high hydrogen production efficiency, the 33HL is considered very suitable for thermophilic H2 fermentation from carbohydrates.

This study demonstrated the H2 or ethanol+H2 production potential by thermophilic dark fermentation. Considering practical applications with the promising thermophilic cultures (AK17 and 33HL), continuous ethanol+H2 or H2 production from pentose sugars and real materials (i.e., organic wastes, lignocellulose hydrolysates) materials should be further studied. In this study, better stability and higher H2 production was obtained by thermophilic dark fermentation processes compared to mesophilic processes. The better stability was related to more stable and less diverse microbial communities in the thermophilic systems compared to mesophilic systems. Further, this study demonstrated ready granulation and high H2 production efficiency of thermophiles, which form basis for further development of thermophilic, high-rate H2 production systems.