New insights into microbial interactions and putative competitive mechanisms during the hydrogen production from tequila vinasses.

This study aimed to characterize the prokaryotic community and putative microbial interactions involved in hydrogen (H2) production during the dark fermentation (DF) process, applying principal components analysis (PCA) to correlate changes in operational, physicochemical, and biological variables. For this purpose, a continuous stirred-tank reactor-type digester fed with tequila vinasses was operated at 24, 18, and 12 h of hydraulic retention times (HRTs) to apply organic loading rates of 20, 36, and 54 g-COD L-1 d-1, corresponding to stages I, II, and III, respectively. Results indicated high population dynamics for Archaea during the DF process toward a decrease in total sequences from 6299 to 99. Concerning the Bacteria community, lactic acid bacteria (LAB) were dominant reaching a relative abundance of 57.67%, while dominant H2-producing bacteria (HPB) decreased from 25.76% to 21.06% during stage III. Putative competitive exclusion mechanisms such as competition for substrates, bacteriocins production, and micronutrient depletion carried out by Archaea and non-H2-producing bacteria (non-HPB), especially LAB, could negatively impact the dominance of HPB such as Ethanoligenens harbinense and Clostridium tyrobutyricum. As a consequence, low maximal volumetric H2 production rate (672 mL-H2 L-1 d-1) and yield (3.88 mol-H2 assimilated sugars-1) were obtained. The global scenario obtained by PCA correlations suggested that C. tyrobutyricum positively impacted H2 molar yield through butyrate fermentation using the butyryl-CoA:acetate CoA transferase pathway, while the most abundant HPB E. harbinense decreased its relative abundance at the shortest HRT toward the dominance of non-HPB. This study provides new insights into the microbial interactions and helps to better understand the DF performance for H2 production using tequila vinasses as substrate. KEY POINTS: • E. harbinense and C. tyrobutyricum were responsible for H2 production. • Clostridiales used acetate and butyrate fermentations for H2 production. • LAB won the competition for sugars against Clostridiales during DF. • Putative bacteriocins production and micronutrients depletion could favor LAB.

Prokaryotic population dynamics and interactions in an AnSBBR using tequila vinasses as substrate in co-digestion with acid hydrolysates of Agave tequilana var. azul bagasse for hydrogen production.

AIMS: The purpose of this study was to characterize the prokaryotic community and putative microbial interactions between H2 -producing bacteria (HPB) and non-HPB using two anaerobic sequencing batch biofilm reactors (AnSBBRs) fed with tequila vinasses in co-digestion with acid hydrolysates of Agave tequilana var. azul bagasse (ATAB). METHODS AND RESULTS: Two AnSBBRs were operated for H2 production to correlate changes in physicochemical and biological variables by principal component analysis (PCA). Results indicated that H2 yield was supported by Ethanoligenens harbinense and Clostridium tyrobutyricum through the PFOR pathway. However, only E. harbinense was able to compete for sugars against non-HPB. Competitive exclusion associated with competition for sugars, depletion of essential trace elements, bacteriocin production and resistance to inhibitory compounds could be carried out by non-HPB, increasing their relative abundances during the dark fermentation (DF) process. CONCLUSIONS: The global scenario obtained by PCA correlated the decrease in H2 production with the lactate:acetate molar ratio in the influent. At the beginning of co-digestion, this ratio had the minimum value considered for a net gain of ATP. This fact could cause the reduction of the relative abundance of C. tyrobutyricum. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that demonstrated the feasibility of H2 production by Clostridiales from acid hydrolysates of ATAB in co-digestion with tequila vinasses.

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 %.

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.

Azospirillum brasilense Increases CO2 Fixation on Microalgae Scenedesmus obliquus, Chlorella vulgaris, and Chlamydomonas reinhardtii Cultured on High CO2 Concentrations.

Mutualism interactions of microalgae with other microorganisms are widely used in several biotechnological processes since symbiotic interaction improves biotechnological capabilities of the microorganisms involved. The interaction of the bacterium Azospirillum brasilense was assessed with three microalgae genus, Scenedesmus, Chlorella, and Chlamydomonas, during CO2 fixation under high CO2 concentrations. The results in this study have demonstrated that A. brasilense maintained a mutualistic interaction with the three microalgae assessed, supported by the metabolic exchange of indole-3-acetic acid (IAA) and tryptophan (Trp), respectively. Besides, CO2 fixation increased, as well as growth and cell compound accumulation, mainly carbohydrates, in each microalgae evaluated, interacting with the bacterium. Overall, these results propose the mutualism interaction of A. brasilense with microalgae for improving biotechnological processes based on microalgae as CO2 capture and their bio-refinery capacity.

Anaerobic treatment of Tequila vinasses in a CSTR-type digester.

Tequila industries in general produce great volumes of effluents with high pollutant loads, which are discharged (untreated or partially treated) into natural receivers, thus causing severe environmental problems. In this contribution, we propose an integrated system as a first step to comply with the Mexican ecological norms and stabilize the anaerobic treatment of Tequila vinasses with main design criteria: simple and easy operation, reduce operating time and associated costs (maintenance), integrated and compact design, minimal cost of set-up, start-up, monitoring and control. This system is composed of a fully instrumented and automated lab-scale CSTR-type digester, on-line measuring devices of key variables (pH, temperature, flow rates, etc.), which are used along with off-line readings of chemical oxygen demand (COD), biogas composition, alkalinity and volatile fatty acids to guarantee the operational stability of the anaerobic digestion process. The system performance was evaluated for 200 days and the experimental results show that even under the influence of load disturbances, it is possible to reduce the COD concentration to 85% in the start-up phase and up to 95% during the normal operation phase while producing a biogas with a methane composition greater than 65%. It is also shown that in order to maintain an efficient treatment, the buffering capacity (given by the alkalinity ratio, alpha = intermediate alkalinity/total alkalinity) must be closely monitored.