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.

Active indole-3-acetic acid biosynthesis by the bacterium Azospirillum brasilense cultured under a biogas atmosphere enables its beneficial association with microalgae.

AIMS: This study assessed, at the physiological and molecular levels, the effect of biogas on indole-3-acetic acid (IAA) biosynthesis by Azospirillum brasilense as well as the impact of this bacterium during CO2 fixation from biogas by Chlorella vulgaris and Scenedesmus obliquus. METHODS AND RESULTS: IpdC gene expression, IAA production and the growth of A. brasilense cultured under air (control) and biogas (treatment) were evaluated. The results demonstrated that A. brasilense had a better growth capacity and IAA production (105.7 ± 10.3 µg ml-1 ) when cultured under biogas composed of 25% CO2  + 75% methane (CH4 ) with respect to the control (72.4 ± 7.9 µg ml-1 ), although the ipdC gene expression level was low under the stressful condition generated by biogas. Moreover, this bacterium was able to induce a higher cell density and CO2 fixation rate from biogas by C. vulgaris (0.27 ± 0.08 g l-1 d-1 ) and S. obliquus (0.22 ± 0.08 g l-1 d-1 ). CONCLUSIONS: This study demonstrated that A. brasilense has the capacity to grow and actively maintain its main microalgal growth-promoting mechanism when cultured under biogas and positively influence CO2 fixation from the biogas of C. vulgaris and S. obliquus. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings broaden research in the field of Azospirillum-microalga interactions and the prevalence of Azospirillum in environmental and ecological topics in addition to supporting the uses of plant growth-promoting bacteria to enhance biotechnological strategies for biogas upgrading.

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.

Biotechnological potential of Chlorella sp. and Scenedesmus sp. microalgae to endure high CO2 and methane concentrations from biogas.

Biogas, a gaseous effluent from the anaerobic digestion of organic waste, is considered an important source of energy, since it has a composition mainly of methane (CH4; 55-75%) and CO2 (20-60%). Today, CO2 from biogas is an excellent carbon source to induce high microalgal biomass production; however, each microalga strain can have different optimal CO2 concentrations for maximizing their bio-refinery capacity as well as different ability to endure stressful conditions of industrial effluents. This study assessed the bio-refinery capacity of Chlorella sp. and Scenedesmus sp., native of Lago de Chapala, Mexico, from biogas, as well as the effect of high CO2 and methane concentrations on the physiological performance to grow, capture CO2 and biochemical composition of both microalgae cultured under different biogas compositions. The results show that both microalgae have the biotechnological potential to endure biogas compositions of 25% CO2-75% CH4. Under this condition, the biomass production attained by Chlorella sp. and Scenedesmus sp. was 1.77 ± 0.32 and 2.25 ± 0.20 g L-1, respectively, with a biochemical composition mainly of carbohydrates and proteins. Overall, this study demonstrates that both microalgae have the ability to endure the stressful biogas composition without affecting their physiological capacity to capture CO2 and biosynthesize high-value metabolites. Moreover, it is worth highlighting the importance of screening wild-type microalgae from local ecosystems to determine their physiological capacity for each biotechnological application.

Agave tequilana bagasse for methane production in batch and sequencing batch reactors: Acid catalyst effect, batch optimization and stability of the semi-continuous process.

Agave tequilana bagasse is the main solid waste of the tequila manufacturing and represents an environmental issue as well as a potential feedstock for biofuel production due to its lignocellulosic composition and abundance. In this contribution, this feedstock was subjected to pretreatments with HCl and H2SO4 for sugar recovery and methane was produced from the hydrolysates in batch and sequencing batch reactors (AnSBR). Sugar recovery was optimized by using central composite designs at different levels of temperature, acid concentration and hydrolysis time. Results showed that at optimal conditions, the HCl pretreatment induced higher sugar recoveries than the H2SO4 one, 0.39 vs. 0.26 g total sugars/g bagasse. Furthermore, the H2SO4 hydrolysate contained higher concentrations of potential inhibitory compounds (furans and acetic acid). Subsequent anaerobic batch assays demonstrated that the HCl hydrolysate is a more suitable substrate for methane production; a four-fold increase was found. A second optimization by using HCl as acid catalyst and methane production as the response variable demonstrated that softer hydrolysis conditions are required to optimize methane production as compared to sugar recovery (1.8% HCl, 119 °C and 103min vs. 1.9% HCl, 130 °C and 133min). This softer conditions were used to feed an AnSBR for 110 days and evaluate its stability at three different cycle times (5, 3 and 2 days). Results showed stable reactor performances at cycle times of 5 and 3 days, obtaining the highest methane yield and production at 3 days, 0.28 NL CH4/g-COD and 1.04 NL CH4/d respectively. Operation at shorter cycle times is not advised due to microbial imbalance.

Nutrient composition of culture media induces different patterns of CO2 fixation from biogas and biomass production by the microalga Scenedesmus obliquus U169.

Microalgae are considered as a promising biotechnological strategy to capture CO2 from biogas, producing biomass with valuable energetic compounds. This study has evaluated the biotechnological potential to fix CO2 from biogas and biomass production by Scenedesmus obliquus cultured in four different formulations of culture media, C30, Bristol, Bold 3N, and C30+M, as well as the effect of methane (CH4) of biogas during CO2 fixation and biomass production. The results obtained showed that different nutrient composition and concentration of culture media, mainly micronutrient and vitamin concentrations, induced different patterns of CO2 fixation from biogas and biomass production in S. obliquus. The highest CO2 fixation (23.03 ± 0.24%) and biomass production (4.63 ± 0.31 g L-1) were attained in the microalgae growing in C30+M medium, accumulating mainly proteins. Moreover, these results demonstrated that the CH4 of biogas did not affect CO2 fixation from biogas and biomass production by S. obliquus. Overall, these results demonstrated that a culture medium with a balanced nutrient supply is crucial to increasing the biotechnological potential of microalgae to fix CO2 from biogas.

CO2 Removal from Biogas by Cyanobacterium Leptolyngbya sp. CChF1 Isolated from the Lake Chapala, Mexico: Optimization of the Temperature and Light Intensity.

In the present study, the capacity of the cyanobacterium Leptolyngbya sp. CChF1 to remove CO2 from real and synthetic biogas was evaluated. The identification of the cyanobacterium, isolated from the lake Chapala, was carried out by means of morphological and molecular analyses, while its potential for CO2 removal from biogas streams was evaluated by kinetic experiments and optimized by a central composite design coupled to a response surface methodology. Results demonstrated that Leptolyngbya sp. CChF1 is able to remove CO2 and grow indistinctly in real or synthetic biogas streams, showing tolerance to high concentrations of CO2 and CH4, 25 and 75%, respectively. The characterization of the biomass composition at the end of the kinetic assays revealed that the main accumulated by-products under both biogas streams were lipids, followed by proteins and carbohydrates. Regarding the optimization experiments, light intensity and temperature were the studied variables, while synthetic biogas was the carbon source. Results showed that light intensity was significant for CO2 capture efficiency (p = 0.0290), while temperature was significant for biomass production (p = 0.0024). The predicted CO2 capture efficiency under optimal conditions (27.1 °C and 920 lx) was 93.48%. Overall, the results of the present study suggest that Leptolyngbya sp. CChF1 is a suitable candidate for biogas upgrading.

Methane production from acid hydrolysates of Agave tequilana bagasse: evaluation of hydrolysis conditions and methane yield.

Evaluation of diluted acid hydrolysis for sugar extraction from cooked and uncooked Agave tequilana bagasse and feasibility of using the hydrolysates as substrate for methane production, with and without nutrient addition, in anaerobic sequencing batch reactors (AnSBR) were studied. Results showed that the hydrolysis over the cooked bagasse was more effective for sugar extraction at the studied conditions. Total sugars concentration in the cooked and uncooked bagasse hydrolysates were 27.9 g/L and 18.7 g/L, respectively. However, 5-hydroxymethylfurfural was detected in the cooked bagasse hydrolysate, and therefore, the uncooked bagasse hydrolysate was selected as substrate for methane production. Interestingly, results showed that the AnSBR operated without nutrient addition obtained a constant methane production (0.26 L CH4/g COD), whereas the AnSBR operated with nutrient addition presented a gradual methane suppression. Molecular analyses suggested that methane suppression in the experiment with nutrient addition was due to a negative effect over the archaeal/bacterial ratio.

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.

Succinic acid production with Actinobacillus succinogenes ZT-130 in the presence of succinic acid.

Glucose fermentation with Actinobacillus succinogenes was carried out at different initial concentrations of succinic acid (SA(0)) to determine its effect on growth and on the production of succinic acid itself. The specific rates of biomass production, succinic, formic and acetic acids decreased with SA(0) (0-40 g/l). The partially dissociated form of succinic acid had a higher effect on cell growth and production of succinic acid as compared to the non-dissociated forms of the acids, a fact that has not been reported until now. Cell growth fitted the Jerusalimski model, and the Aiba-Shoda model was suitable for quantification of the inhibition for the production of succinic acid. The growth inhibition constants K(is) and K(ip) and their summatory were consistent with the experimental values obtained, i.e., 22 g/l for the produced acids and 38 g/l for total acids that were the limits at which the biomass formation ceased.

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis.

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.