Knowing the enemy: homoacetogens in hydrogen production reactors.

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors. KEY POINTS: • qPCR and sequencing analysis revealed two homoacetogenesis phenomena. • fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.

Continuous thermophilic hydrogen production from an enzymatic hydrolysate of agave bagasse: Inoculum origin, homoacetogenesis and microbial community analysis.

In this research, the performance of two thermophilic inocula of different origin on continuous hydrogen production from an enzymatic hydrolysate of agave bagasse were compared; one of them was obtained from a thermophilic reactor and the second one was taken from a mesophilic reactor and acclimated to thermophilic conditions. The acclimation process in one-step quickly established a high-performance hydrogen producing community, obtaining a volumetric hydrogen production rate of 3811 ± 19 mL H2/L-d with an hydrogen yield of 121 L H2/kg bagasse compared to 1473 ± 6 mL H2/L-d and 26.6 L H2/kg obtained with the thermophilic-origin inoculum. The differences in the performance of both inocula were closely linked to the profile of volatile fatty acids produced, the homoacetogenic pathway and the microbial community, the latter being the determining factor. The use of mesophilic-origin inoculum acclimated to thermophilic conditions can significantly improve the hydrogen production from lignocellulosic bagasse.

Standardized protocol for determination of biohydrogen potential.

Biohydrogen production potential (BHP) depends on several factors like inoculum source, substrate, pH, among many others. Batch assays are the most common strategy to evaluate such parameters, where the comparison is a challenging task due to the different procedures used. The present method introduces the first internationally validated protocol, evaluated by 8 independent laboratories from 5 different countries, to assess the biohydrogen potential. As quality criteria, a coefficient of variation of the cumulative hydrogen production (H max) was defined to be <15 %. Two options to run BHP batch tests were proposed; a manual protocol with periodic measurements of biogas production, needing conventional laboratory materials and analytical equipment for biogas characterization; and an automatic protocol, which is run in a device developed for online measurements of low biogas production. The detailed procedures for both protocol options are presented, as well as data validating them. The validation showed acceptable repeatability and reproducibility, measured as intra- and inter-laboratory coefficient of variation, which can be reduced up to 9 %.

Improvement of continuous hydrogen production using individual and binary enzymatic hydrolysates of agave bagasse in suspended-culture and biofilm reactors.

Continuous hydrogen (H2) production from individual (Stonezyme, IH) and binary (Celluclast-Viscozyme, BH) enzymatic hydrolysates of agave bagasse was evaluated in continuous stirred-tank reactors (CSTR) and trickling bed reactors (TBR). The volumetric H2 production rates (VHPR) in CSTR were 13 and 2.25 L H2/L-d with BH and IH, respectively. Meanwhile, VHPR of 5.76 and 2.0 L H2/L-d were obtained in the TBR configuration using BH and IH, respectively. Differences on VHPR between reactors could be explained by substrate availability, which is intrinsic to the growth mode of each reactor configuration; while differences of VHPR between hydrolysates were possibly related to the composition of enzymatic hydrolysates. Furthermore, homoacetogenesis was strongly influenced by H2 and substrate transfer conditions. Considering VHPR, H2 yields, and costs of hydrolysis, hydrogen production from binary hydrolysates of agave bagasse was identified as the most promising alternative evaluated with scale-up potential for the production of energy biofuels.

Microbial contamination in methanol biofilters inoculated with a pure strain of Pichia pastoris: A potential limitation for waste revalorization.

Novel biotechnologies to valorize waste emissions are based on the use of specialized microbial groups that produce different compounds of industrial interest. On this scenario, the retention of such specific microorganisms in the system is of critical interest; however, the potential limitations of working with simplified cultures in a competitive open environment are neither fully explored nor well understood. In this work, a series of biofilters treating methanol vapors coupled with heterologous endochitinase production were used to evaluate the performance of a specialized microbial population during a typical open-to-environment operation. The biofilters were inoculated with a transformed strain of Pichia pastoris and were operated identically for about 90 days. The results showed that the biofiltration performance became diverse with time in terms of the elimination capacity (EC) shifting from a variation coefficient of 1.5% (EC = 274 ± 24, 279 ± 5, and 281.9 ± 25 g/[m3 h]) at the beginning of the operation to 33% (EC = 297 ± 9, 338 ± 7, and 341 ± 2 g/[m3 h]) at the end of operation. Epifluorescence analysis and cloning-sequencing suggested that P. pastoris remained as the dominant microorganism of methanol degradation, whereas diverse airborne bacteria, including Ochrobactrum spp. and Klebsiella oxytoca, played a secondary role possibly associated with the consumption of intermediates. Overall, this study found that low diversity systems operated under non-sterile conditions could be susceptible to contamination with external microorganisms causing a diversifying behavior at the performance and microbial community levels. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2715, 2019.

Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor.

The hydrogen (H2) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the selection of microbial species that either consume molecular H2 or outcompete hydrogenogenic bacteria for the organic substrate. In this study, the effect of organic loading rate (OLR) on the H2 production performance, the metabolic pathways, and the microbial community composition in a continuous system was evaluated. Two bacterial genera, Clostridium and Streptococcus, were dominant in the microbial community depending on the OLR applied. At low OLR (14.7-44.1 gLactose/L-d), Clostridium sp. was dominant and directed the system towards the acetate-butyrate fermentation pathway, with a maximum H2 yield of 2.14 molH2/molHexose obtained at 29.4 gLactose/L-d. Under such conditions, the volumetric hydrogen production rate (VHPR) was between 3.2 and 11.6 LH2/L-d. In contrast, relatively high OLR (58.8 and 88.2 gLactose/L-d) favored the dominance of Streptococcus sp. as co-dominant microorganism leading to lactate production. Under these conditions, the formate production was also stimulated serving as a strategy to dispose the surplus of reduced molecules (e.g., NADH2+), which theoretically consumed up to 5.72 LH2/L-d. In such scenario, the VHPR was enhanced (13.7-14.5 LH2/L-d) but the H2 yield dropped to a minimum of 0.74 molH2/molHexose at OLR = 58.8 gLactose/L-d. Overall, this research brings clear evidence of the intrinsic occurrence of metabolic pathways detrimental for biohydrogen production, i.e., lactic acid fermentation and formate production, suggesting the use of low OLR as a strategy to control them.