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Colombia's continuous contamination of water resources and the low alternatives to produce biofuels have affected the fulfillment of the objectives of sustainable development, deteriorating the environment and affecting the economic productivity of this country. Due to this reality, projects on environmental and economic sustainability, phytoremediation, and the production of biofuels such as ethanol and hydrogen were combined. The objective of this article was to design and develop a sustainable system for wastewater treatment and the generation of biofuels based on the biomass of the aquatic plant Eichhornia crassipes. A system that simulates an artificial wetland with live E. crassipes plants was designed and developed, removing organic matter contaminants; subsequently, and continuing the sustainability project, bioreactors were designed, adapted, and started up to produce bioethanol and biohydrogen with the hydrolyzed biomass used in the phytoremediation process, generating around 12 g/L of bioethanol and around 81 ml H2/g. The proposed research strategy suggests combining two sustainable methods, bioremediation and biofuel production, to preserve the natural beauty of water systems and their surroundings.

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Water scarcity is a significant global issue caused by the prolonged disregard and unsustainable management of this essential resource by both public and private bodies. The dependence on fossil fuels further exacerbates society's bleak environmental conditions. Therefore, it is crucial to explore alternative solutions to preserve our nation's water resources properly and promote the production of biofuels. Research into the utilization of E. crassipes to remove heavy metals and generate biofuels is extensive. The combination of these two lines of inquiry presents an excellent opportunity to achieve sustainable development goals. This study aims to develop a sustainable wastewater treatment system and generate biohydrogen from dry, pulverized E. crassipes biomass. A treatment system was implemented to treat 1 L of industrial waste. The interconnected compartment system was built by utilizing recycled PET bottles to generate biohydrogen by reusing the feedstock for the treatment process. The production of biological hydrogen through dark fermentation, using biomass containing heavy metals as a biohydrogen source, was studied. Cr (VI) and Pb (II) levels had a low impact on hydrogen production. The uncontaminated biomass of E. crassipes displayed a significantly higher hydrogen yield (81.7 mL H2/g glucose). The presence of Cr (IV) in E. crassipes leads to a decrease in biohydrogen yield by 14%, and the presence of Pb (II) in E. crassipes leads to a decrease in biohydrogen yield of 26%. This work proposes a strategy that utilizes green technologies to recover and utilize contaminated water. Additionally, it enables the production of bioenergy with high efficiency, indirectly reducing greenhouse gases. This strategy aligns with international programs for the development of a circular economy.

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This research aimed at evaluating optimal conditions to obtain value-added metabolites, such as bio-CH4, by co-digesting swine manure and food waste diluted in domestic sewage. The assays were carried out in batches using the statistical methods of Rotational Central Composite Design (RCCD) and Surface Response to evaluate the ranges of food waste (1.30-9.70 gTS.L-1), pH (6.16-7.84) and granular Upflow Anaerobic Sludge Blanket sludge as inoculum (2.32-5.68 gTS.L-1), besides about 250 mL of swine manure in 500 mL Duran flasks. According to the RCCD matrix, bio-CH4 yields among 600.6 ± 60.1 and 2790.0 ± 112.0 mL CH4 gTS.L-1 were observed, besides the maximum CH4 production rate between 0.4 ± 0.5 and 49.7 ± 2.0 mL CH4 h-1 and λ between ≤0.0 and 299.3 ± 4.5 h. In the validation assay, the optimal conditions of 9.98 gTS.L-1 of food waste, pH adjusted to 8.0 and 2.20 gTS.L-1 of inoculum were considered, and the bio-CH4 yield obtained (5640.79 ± 242.98 mL CH4 gTS.L-1 or also 5201.83 ± 224.07 mL CH4 gTVS.L-1) was 11.3 times higher than in assays before optimization (499.3 ± 16.0 mL CH4 gTS.L-1) with 5 gTS.L-1 of food waste, 3 gTS.L-1 of inoculum and pH 7.0. Besides, the results observed about the energetic balance of the control and validation assays highlight the importance of process optimization, as this condition was the only one with energy supply higher than the energy required for its operation, exceeding max consumption sevenfold. Based on the most dominant microorganisms (Methanosaeta, 31.06%) and the metabolic inference of the validation assay, it could be inferred that the acetoclastic methanogenesis was the predominant pathway to CH4 production.

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An alternative to improve the production of biorefinery products, such as biohydrogen (H2) and volatile fatty acids (VFA), is the combination of nanotechnology and biological processes. In order to compare the use of both processes in two different reactor configurations, batch reactors and continuous anaerobic fluidized bed reactors (AFBR) were studied under the same conditions (37°C, pH 6.8, Clostridium butyricum as an inoculum and glucose as a substrate) to evaluate the influence of zero valence iron and nickel nanoparticles (NPs) on H2 and VFA production. There was a shift in the production of acetic and butyric acids to produce mainly valeric acid when NPs were added in batch reactors. Meanwhile, in AFBR the change was from lactic acid to butyric and acetic acids with the addition of NPs. It showed that the effect of NPs on the fermentation process was different when the configuration of batch and continuous reactors was compared. The H2 yield in both reactor configurations increased with the addition of NPs. In batch reactors from 6.6 to 8.0 mmol H2 g-1 of COD and in AFBR from 4.9 to 6.2 mmol of H2 g-1 of COD. Therefore, given the simplicity and low cost of the synthesis of metallic NPs, it is a promising additive to optimize the fermentation process in different reactor configurations.

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Considering the scarcity of data in the literature regarding phylogenetic and metabolic composition of different inocula, especially those from thermophilic conditions, this research aimed at characterizing the microbial community and preferable metabolic pathways of an UASB reactor sludge applied to the thermophilic treatment (55°C) of sugarcane vinasse, by means of shotgun metagenomics. After its metabolic potential was depicted, it was possible to observe several genes encoding enzymes that are of great importance to anaerobic digestion processes with different wastes as substrate, especially regarding the biodegradation of carbohydrates and ligninolytic compounds, glycerolypids, volatile fatty acids and alcohols metabolism and biogas (H2 and CH4) production. The genera identified in higher relative abundances for Bacteria domain were Sulfirimonas (37.52 ± 1.8%), possibly related to the sludge endogenic activity due to its strong relation with a peptidoglycan lyase enzymes family, followed by Fluviicola (5.01 ± 1.0%), Defluviitoga (4.36 ± 0.2%), Coprothermobacter (4.32 ± 0.5%), Fervidobacterium (2.93 ± 0.3%), Marinospirillum (2.75 ± 0.2%), Pseudomonas (2.14 ± 0.2%) and Flavobacterium (1.78 ± 0.1%), mostly related with carbohydrates fermentations and/or H2 production. For Archaea domain, Methanosarcina (0.61 ± 0.1%), Methanothermobacter (0.38 ± 0.0%), Methanoculleus (0.30 ± 0.1%), Thermococcus (0.03 ± 0.0%), Methanolobus (0.02 ± 1.8%), Methanobacterium (0.013 ± 0.0%), Aciduliprofundum and Pyrococcus (0.01 ± 0.0%) were the most dominant ones, being Methanosarcina the most related with methanogenesis. It was concluded that the robust inoculum description performed in this study may subside future biotechnological researches by using similar inocula (UASB sludges), focusing on the obtainment of value-added by-products by means of anaerobic digestion, such as volatile fatty acids, alcohols and biogas (H2 and CH4), by using several types of waste as substrate.

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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.

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The increase in biofuel production by 2030, driven by the targets set at the 21st United Nations Framework Convention on Climate Change (COP21), will promote an increase in ethanol production, and consequently more vinasse generation. Sugarcane vinasse, despite having a high polluting potential due to its high concentration of organic matter and nutrients, has the potential to produce value-added resources such as volatile fatty acids (VFA), biohydrogen (bioH2) and biomethane (bioCH4) from anaerobic digestion. The objective of this paper is to present a critical review on the vinasse treatment by anaerobic digestion focusing on the final products. Effects of operational parameters on production and recovery of these resources, such as pH, temperature, retention time and type of inoculum were addressed. Given the importance of treating sugarcane vinasse due to its complex composition and high volume generated in the ethanol production process, this is the first review that evaluates the production of VFAs, bioH2 and bioCH4 in the treatment of this organic residue. Also, the challenges of the simultaneous production of VFA, bioH2 and bioCH4 and resources recovery in the wastewater streams generated in flex-fuel plants, using sugarcane and corn as raw material in ethanol production, are presented. The installation of flex-fuel plants was briefly discussed, with the main impacts on the treatment process of these effluents either jointly or simultaneously, depending on the harvest season.

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This work characterizes and comparatively assess two cation exchange membranes (PSEBS SU22 and CF22 R14) and one bipolar membrane (FBM) in microbial electrolysis cells (MEC), fed either by acetate or the mixture of volatile fatty acids as substrates. The PSEBS SU22 is a new, patent-pending material, while the CF22 R14 and FBM are developmental and commercialized products. Based on the various MEC performance measures, membranes were ranked by the EXPROM-2 method to reveal which of the polymeric membranes could be more beneficial from a complex, H2 production efficiency viewpoint. It turned out that the substrate-type influenced the application potential of the membranes. Still, in total, the PSEBS SU22 was found competitive with the other alternative materials. The evaluation of MEC was also supported by analyzing anodic biofilms following electroactive bacteria's development over time.

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The use of effluents for hydrogen production through dark fermentation is promising because it results in the generation of value-added products and reduction of the effluent's organic load. A low-cost medium using agroindustrial effluents, corn steep liquor (CSL) and cassava processing wastewater (CPW) was evaluated for hydrogen production with microbial consortia (Vir and Gal). Four variables were evaluated for their impact on biohydrogen production through a Plackett Burman design. Subsequently, the significant variables were optimized using a central composite design, resulting in two mathematical models with regression coefficients R2 > 0.92. The maximum yields were validated and resulted in 107 and 83.1 mL of biohydrogen/g COD removed for Vir and Gal, respectively. The lower medium cost for biohydrogen production was 81.5 USD/m3, approximately 80% more economical than some supplemented media. Finally, the scale-up of the biohydrogen production by consortia to 5L resulted in an increase of more than 40%.

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In this work, lawsone (LQ) and anthraquinone 2-sulphonate (AQS) (dissolved and covalently immobilized on activated carbon) were evaluated as redox mediators during the dark fermentation of glucose by a pretreated anaerobic sludge. Findings revealed that the use of dissolved LQ increased H2 production (10%), and dissolved AQS improved H2 production rate (11.4%). Furthermore, the total production of liquid byproducts (acetate, butyrate, ethanol, and butanol) was enhanced using dissolved (17%) and immobilized (36%) redox mediators. The established redox standard potentials of LQ and AQS suggested a possible interaction through electron transfer in cytochromes complexes for hydrogen production and the Bcd/EtfAB complex for volatile fatty acid formation.

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Palm oil mill effluent (POME) was tested as a substrate to produce hydrogen by dark fermentation. Two microbial consortia and a pure culture of Clostridium beijerinckii (ATCC 8260) were cultured anaerobically in raw, diluted and hydrolyzed POME to compare biohydrogen production yields in all three media. Experiments were done in 15 mL Hungate tubes containing 5 mL of medium and 1 mL of inoculum. When Clostridium beijerinckii was cultivated at 30 °C in the hydrolyzed POME (P003), containing 7.5 g/L of sucrose, during 8 days of fermentation and 20 % of the inoculum, the maximum biohydrogen production yield was 4.62 LH2/Lmed. Consortium C3 also showed the best production in hydrolyzed POME while consortium C6 achieved its maximum production in raw POME. This effluent is a potential substrate for biohydrogen production.

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Renewable energy can assist the management of the effects of population growth and rapid economic development on the sustainability of animal husbandry. The primary aim of renewable energy is to minimize the use of fossil fuels via the creation of environmentally friendly energy products from depleted fossil fuels. Digesters that treat swine manure are extensively used in treatment systems; and inclusion of swine carcasses can increase the organic loading rate (OLR) thereby improving biogas yield and productivity on farms. However, the characteristics of the components including animal residues, proteins, lipids, remains of undigested feed items, antimicrobial drug residues, pathogenic microorganisms and nutrient contents, are complex and diverse. It is therefore necessary to manage the anaerobic process stability and digestate purification for subsequent use as fertilizer. Efficient methane recovery from residues rich in lipids is difficult because such residues are only slowly biodegradable. Pretreatment can promote solubilization of lipids and accelerate anaerobic digestion, and pretreatments can process the swine carcass before its introduction onto biodigesters. This review presents an overview of the anaerobic digestion of swine manure and carcasses. We analyze the characteristics of these residues, and we identify strategies to enhance biogas yield and process stability. We consider energy potential, co-digestion of swine manure and carcasses, physical, chemical, and biological pretreatment of biomass, sanitary aspects of swine manure and co-digestates and their recycling as fertilizers.

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Biohydrogen production was evaluated using cassava processing wastewater (CPW) and two microbial consortia (Vir and Gal) from different Brazilian environments. The biohydrogen production was optimized using a Box-Behnken design (T, pH, C/N, and % v/v inoculum). Maximum yields were obtained with hydrolyzed substrate: 4.12 and 3.80 mol H2 / for Vir and Gal, respectively. Similarly, the kinetic parameters µ, k, and q were higher with hydrolyzed CPW in both consortia. The molecular analysis of the consortia through Illumina high-throughput sequencing showed the presence of bacteria from the families Porphyromonadaceae, Clostridiaceae, Ruminococcaceae, and Enterococcaceae. The relative abundance of microbial families varies as fermentation progresses. In both consortia, Clostridiaceae reached the maximum relative abundance in the media between 16 and 24 h, interval in which approximately 90% of the biohydrogen is generated.

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In this work, two commercialized anion-exchange membranes (AEMs), AMI-7001 and AF49R27, were applied in microbial electrolysis cells (MECs) and compared with a novel AEM (PSEBS CM DBC, functionalized with 1,4-diazabicyclo[2.2.2]octane) to produce biohydrogen. The evaluation regarding the effect of using different AEMs was carried out using simple (acetate) and complex (mixture of acetate, butyrate and propionate to mimic dark fermentation effluent) substrates. The MECs equipped with various AEMs were assessed based on their electrochemical efficiencies, H2 generation capacities and the composition of anodic biofilm communities. pH imbalances, ionic losses and cathodic overpotentials were taken into consideration together with changes to substantial AEM properties (particularly ion-exchange capacity, ionic conductivity, area- and specific resistances) before and after AEMs were applied in the process to describe their potential impact on the behavior of MECs. It was concluded that the MECs which employed the PSEBS CM DBC membrane provided the highest H2 yield and lowest internal losses compared to the two other separators. Therefore, it has the potential to improve MECs.

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Conversion of organic matter to biohydrogen possesses promising application potential. In this study, low-cost ferrihydrite nanorods were used to enhance hydrogen production by Clostridium pasteurianum. The maximum cumulative hydrogen production and the hydrogen yield were 1.03 mmol and 3.55 mol H2/mol glucose, respectively, which were 68.9% and 15.6% higher than those of the batch groups without ferrihydrite addition. Moreover, in comparison with magnetite and hematite nanoparticles, ferrihydrite presented the best stimulation for hydrogen evolution. The enhancement mechanisms were explored based on metabolic distribution, gene expression, enzymatic activity, and metabolite determination, such as Fe(II) concentration and pH value. The potential stimulation mechanisms are summarized as follows: ferrihydrite improves glucose conversion efficiency and promotes cell growth; ferrihydrite elevates the transcripts and activity of hydrogenase; and ferrihydrite reduction via its buffer function could ease acidification. This study demonstrates that ferrihydrite addition is an effective and green strategy to enhance fermentative hydrogen production.

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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.

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This research work has succeeded in recovering energy from glucose by generating H2 with the aid of a Clostridium beijerinckii strain and obtaining electrical energy from compounds present in the H2 fermentation effluent in a microbial fuel cell (MFC) seeded with native port drainage sediment. In the fermentation step, 49.5% of the initial glucose concentration (56 mmol/L) was used to produce 104 mmol/L H2; 5, 33, 3, and 1 mmol/L acetate, butyrate, lactate, and ethanol also emerged, respectively. MFC tests by feeding the anodic compartment with acetate, butyrate, lactate (individually or as a mixture), or the H2 fermentation effluent provided power density values ranging between 0.6 and 1.2 W/m2. Acetate furnished the highest power density with a nanowire-rich biofilm despite the lowest anode bacterial concentration (1012 16S gene copies/g of sediment). Non-conventional exoelectrogenic microbial communities were observed in the acetate-fed MFC; e.g., Pseudomonadaceae (Pseudomonas) and Clostridia (Acidaminobacter, Fusibacter).

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Winery wastewaters are acidic effluents with high content of organic matter and nutrients. Different initial values of chemical oxygen demand (COD), ranging from 4 to 50 g L-1, were tested in batch assays to evaluate the fermentative hydrogen production followed by a methane production step. The influence of adding a typical nutrient solution for hydrogen production was investigated. Nutrients include N-NH4, Mg, Fe, Co, Mn, I, Ni, and Zn. The best hydrogen production potential was obtained at a COD of 50 g L-1 without nutrient addition. This condition produced 528 mL H2 L-1. At a COD ≥ 35 g L-1, tests with only WW had a hydrogen potential 1.6 to 1.9 times higher than did tests where nutrients were added. The use of added nutrients reduced the hydrogen production by producing additional reduced acids, such as propionate and valerate. In a second stage, biomethane potential was evaluated using the effluent of a selected condition from hydrogen production tests. The methane production reached values of 207 ± 2.2 mL CH4 g-1 COD at 10 g COD L-1. The COD affected the specific methane production. The results of this study demonstrated the potential of winery effluents as a substrate for sequential hydrogen and methane production to increase the energy recovery from this effluent, with a maximum energetic yield and productivity of 7.15 kJ gCOD-1 and 11.51 kJ d-1.

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AIM: The aim of this research was to create a work scheme for the isolation of the different micro-organisms commonly found in hydrogen-producing reactors and to test its effectiveness. METHODS AND RESULTS: Methods were selected to isolate anaerobic spore-forming fermenters, anaerobic fermenters that do not form spores, facultative aerobic fermenters and lactic acid bacteria. The methods were tested in two samples taken from a hydrogen-producing reactor fed with cheese whey. 16S rRNA gene sequences from isolates were compared with pyrosequencing analysis from the same samples. The isolates represented more than 88% of the abundance detected by pyrosequencing. Organisms from the genera Clostridium, Rahnella, Megasphaera, Lactobacillus, Propionibacterium, Bifidobacterium, Chryseobacterium and Acetobacter were isolated. Hydrogen-producing capacity was confirmed for the Clostridium, Rahnella and Megasphaera isolates. Coculture experiments indicate that Megasphaera prevented the total inhibition of Clostridium by Lactobacillus. CONCLUSION: The work scheme proposed was effective to isolate most of the micro-organisms detected by pyrosequencing analysis. Physiological studies suggested a key role of Megasphaera. SIGNIFICANCE AND IMPACT OF THE STUDY: We showed the high culturability of the microbial communities from hydrogen-producing bioreactors. The isolates can be used to perform physiological studies to understand the H2 -producing process.

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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.

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