Evaluation of hydrogen fermentation by a newly isolated alkaline tolerant Clostridium felsineum strain CUEA03

A new alkaline tolerant Clostridium felsineum strain (CUEA03) was isolated from a mangrove sediment in Thailand. Genomics revealed that this strain contained a 5,081,113 bp genome sequence with 4797 predicted protein coding sequences, comprised of a large number of genes that are linked to several processes of carbon utilization, hydrogen (H₂), and butanol production. Chemical tests revealed that the bacteria have a high potential for utilization of a wide range of carbon sources. After optimization by batch fermentation, a maximum cumulative H₂ production (CHP) of 5425 mL/L in 72 h was obtained at pH 9, 30 °C, 0.9 g/L NaCl, and 35 g/L glucose. Moreover, this strain was compatible with agro-industrial waste, giving a CHP of 5187 mL/L (893 mL/g COD_consumed). This demonstrates that environmentally isolated organisms have the potential to be used as robust high H₂ producers from various complex organic substrates.     

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h^?1), a higher butyric acid production of 6.8 g·L^?1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h^?1, with a constant hydrogen production of 507 mL·h^?1, corresponding to a volumetric rate of 6.10 L·L^?1 d^?1, and a yield of 2.39 mol of H₂ per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H₂/g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production.     

Harvesting biohydrogen from toxic wastewater using isolated strain

Toxicity prevents the bioenergy content of certain industrial effluents from being recovered. An enriched Clostridium butyricum strain was employed to produce hydrogen by fermentation from cellobiose in the presence of phenol at 200–1500 mgl⁻¹. The enriched Cl. butyricum yielded the most hydrogen at 2.1 mol H₂ mol⁻¹ cellobiose with 600 mgl⁻¹ phenol. Butyrate was the main metabolite. Cell metabolism was substantially inhibited at a phenol concentration of 1500 mgl⁻¹. Part of the phenol was co-degraded during the test, helping to eliminate the toxicity of wastewater. Both the pyruvate oxidative decarboxylation pathway and the NADH pathway contributed to biohydrogen production. Phenol toxicity more strongly inhibits soluble hydrogenase than it does membrane-bound hydrogenase. Although the NADH pathway dominated at low phenol concentration, increasing the phenol concentration shifted the biohydrogen pathway toward decarboxylation.     

The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process

In view of increasing attempts for the production of renewable energy, the production of biohydrogen energy by a new mesophilic bacterium Clostridium sp. YM1 was performed for the first time in the dark fermentation. Experimental results showed that the fermentative hydrogen was successfully produced by Clostridium sp. YM1 with the highest cumulative hydrogen volume of 3821 ml/L with a hydrogen yield of 1.7 mol H₂/mol glucose consumed. Similar results revealed that optimum incubation temperature and pH value of culture medium were 37 °C and 6.5, respectively. The study of hydrogen production from glucose and xylose revealed that this strain was able to generate higher hydrogen from glucose compared to that from xylose. The profile of volatile fatty acids produced showed that hydrogen generation by Clostridium sp. YM1 was butyrate-type fermentation. Moreover, the findings of this study indicated that an increase in head space of fermentation culture positively enhanced hydrogen production.     

Enhanced biohydrogen production from date seeds by Clostridium thermocellum ATCC 27405

Biohydrogen production from waste lignocellulosic biomass serves the dual purpose of converting waste into valuable products and alleviates waste disposal issues. In this study, waste date seeds were valorized for biohydrogen production via consolidated bioprocessing by Clostridium thermocellum ATCC 27405. Effect of various surfactants (PEG1000, surfactin, Triton X-100) and sodium carbonate (buffering agent) on biohydrogen production from the acid pre-treated substrate was examined. Among the various surfactants, addition of Triton X-100 resulted in the maximum biohydrogen yield of 103.97 mmol/L at an optimal dosage of 0.75% w/v. Triton X-100 supplementation favoured the production of ethanol and acetate as co-metabolites than butyrate. Addition of Na₂CO₃ to date seed fermentation medium at a concentration of 15 mM enhanced the biohydrogen production by 33.16%. Also, Na₂CO₃ buffering supported the glycolytic pathway and subsequent ethanol production than acetate/butyrate formation. Combined effect of the optimal dosages of Triton X-100 and Na₂CO₃ resulted in high hydrogen productivity up to 72 h (0.443 mmol/g h of H₂) with a total increase in hydrogen yield of 40.6% at the end of 168 h, as compared to fermentation supplemented with Triton X-100 alone. Further analysis revealed that the combined effects of the additives resulted in better substrate degradation, favourable pH window and cell growth promotion which ensured enhanced hydrogen productivity and yield. Thus, the study highlights a novel stimulatory approach for enhanced biohydrogen production from a new substrate.     

Optimization of biohydrogen production by the novel psychrophilic strain N92 collected from the Antarctica

In this study, the response surface methodology (RSM) with central composite design (CCD) was employed to improve the hydrogen production by the psychrophilic N92 strain (EU636058) isolated from Antarctica, which is closely related to Pseudorhodobacter sp. (KT163920). The influence of operational conditions such as temperature (4.7–55.2 °C), initial pH (3.44–10.16), and initial glucose concentration (4.7–55.23 g/dm³), as well as the initial concentrations of (NH₄)₂SO₄ (0.05–3.98 g/dm³), FeSO₄ (0.02–1.33 g/dm³) and NaHCO₃ (0.02–3.95 g/dm³) was evaluated. The linear effect of glucose concentration, along with the quadratic effect of all the six factors were the most significant terms affecting the biohydrogen yield by N92 strain. The optimum conditions for the maximum hydrogen yield of 1.7 mol H₂/mol glucose were initial pH of 6.86, glucose concentration of 28.4 g/dm³, temperature 29 °C and initial concentration of (NH₄)₂SO₄, FeSO₄ and NaHCO₃ of 0.53, 1.55 and 1.64 g/dm³ respectively. Analysis of the metabolites produced under the optimum conditions showed that the most abundant were acetic acid (0.8 g/dm³), butyric acid (0.7 g/dm³) and ethanol (2.1 g/dm³). We suggest that the bioprocess established in this study using the strain N92 could be an alternative for hydrogen production with the advantages of constituting low energy costs in fermentation.     

Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor

To achieve stable and efficient photo-fermentative hydrogen production, this work investigated photo-fermentative hydrogen production by forming biofilm on the surface of carrier in the biofilm reactor (BR). Results showed the hydrogen production performance was greatly improved by formed biofilm. The time of hydrogen production and efficiency of substrate utilization were enhanced obviously compared to the control reactor (CR). When the CR was used, hydrogen production stopped at 7th day and maximum cumulative hydrogen volume and hydrogen yield were 1730 ± 87 mL/L and 1.44 ± 0.07 mol H₂/mol acetate, respectively. However, in the BR hydrogen production volume of 3028 ± 150 mL/L and hydrogen yield of 2.52 ± 0.13 mol H₂/mol acetate were obtained, which were enhanced about 75% compared to that of the CR. The time of hydrogen production extended from 7 days of CR to 12 days of BR and the substrate conversion efficiency increased from 36% of CR to 63% of BR. It was worth noting at 8th day that substrate was almost utilized completely but hydrogen production still lasted for 4 days. This suggested that the formation of biofilm in BR was favorable to continuous hydrogen production and substrate utilization with high efficiency. Results demonstrated the BR can get a more stable and consistent operating process and it was a proper and potential way to produce hydrogen by photo-fermentative bacteria (PFB).     

The CRISPR technology: A promising strategy for improving dark fermentative biohydrogen production using Clostridium spp.

Generating hydrogen gas (H₂) using the dark fermentation method has attracted much attention due to its lower energy requirement and environmental friendliness. However, producing a high yield of bio-H₂ is as challenging as ever due to low energy conversion by microorganisms. In this respect, the advancement of genome editing tools including the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology could overcome the established maximum ceiling of product yield. To date, CRISPR-Cas systems, particularly those based on Type II CRISPR-Cas9 and Type V CRISPR-Cas12, are widely used in manipulating novel bacteria to improve the yield of specific biofuel. However, studies using the CRISPR-Cas technology for improving bio-H₂ production remain scarce. Understanding the metabolic pathways of Clostridium spp. is essential for using the CRISPR-Cas technology Thus, this review highlighted the state-of-the-art in CRISPR-Cas systems for bacterial genome editing while paying attention to bioprocess optimization strategies for modulating the biohydrogen production.     

High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant

A mesophilic high hydrogen producing strain DMHC-10 was isolated from a lab scale anaerobic reactor being operated on distillery wastewater for hydrogen production. DMHC-10 was identified as Clostridium sp. on the basis of 16S rRNA gene sequencing. Various medium components (carbon and nitrogen sources) and environmental factors (initial pH, temperature of incubation) were optimized for hydrogen production by Clostridium sp. DMHC-10. The strain, in late exponential growth phase, showed maximum hydrogen production (3.35 mol-H₂ mol⁻¹ glucose utilized) at 37 °C, pH 5.0 in a medium supplemented with organic nitrogen source. Butyric acid to acetic acid ratio was ca. 2.3. Hydrogen production declined when organic nitrogen was replaced with inorganic nitrogen.     

Fermentative biohydrogen production from starch-containing textile wastewater

The present study deals with the biohydrogen production from starch-containing wastewater collected from the textile industry in Taiwan. The effects of inoculums collected from different sources (sewage sludge, soil and cow dung), substrate concentrations (5–25 g COD/L) and pH (4.0–8.0) on hydrogen production from wastewater were investigated.     

Improvement of biohydrogen production from glycerol in micro-oxidative environment

Glycerol is a highly available by-product generated in the biodiesel industry. It can be converted into higher value products such as hydrogen using biological processes. The aim of this study was to optimize a continuous dark fermenter producing hydrogen from glycerol, by using micro-aerobic conditions to promote facultative anaerobes. For that, hydrogen peroxide (H₂O₂) was continuously added at low but constant flow rate (0.252 mL/min) with three different inlet concentrations (0.2, 0.4, and 0.6% w/w). A mixture of aerobic and anaerobic sludge was used as inoculum. Results showed that micro-oxidative environment significantly enhanced the overall hydrogen production. The maximum H₂ yield (403.6 ± 94.7 mmolH₂/molGly_consumed) was reached at a H₂O₂ concentration of 0.6% (w/w), through the formate, ethanol and butyrate metabolic pathways. The addition of H₂O₂ promoted the development of facultative anaerobic microorganisms such as Klebsiella, Escherichia-Shigella and Enterococcus sp., likely by consuming oxygen traces in the medium and also producing hydrogen. Despite the micro-oxidative environment, strict anaerobes (Clostridium sp.) were still dominant in the microbial community and were probably the main hydrogen producing species. In conclusion, such micro-oxidative environment can improve hydrogen production by selecting specific microbial community structures with efficient metabolic pathways.     

Optimization of biohydrogen production of palm oil mill effluent by ozone pretreatment

The biohydrogen (H₂) production in batch experiments under varying concentrations of raw and ozonated palm oil mill effluent (POME) of 5000–30,000 mg COD.L⁻¹, at initial pH 6, under mesophilic (37 °C), thermophilic (55 °C) and extreme-thermophilic (70 °C) conditions. Effects of ozone pretreatment, substrate concentration and fermentation temperature on H₂ production using mesophilic seed sludge was undertaken. The results demonstrated that H₂ can be produced from both raw and ozonated POME, and the amounts of H₂ production were directly increased as the POME concentrations were increased. H₂ was successfully produced under the mesophilic fermentation of ozonated POME, with maximum H₂ yield, and specific H₂ production rate of 182 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 6.2 mL.h⁻¹.g⁻¹ TVS (25,000 mg COD.L⁻¹), respectively. Thus, indicating that the ozone pretreatment could elevate on the biodegradability of major constituents of the POME, which significantly enhanced yields and rates of the H₂ production. H₂ production was not achieved under the thermophilic and extreme-thermophilic fermentation. In both fermentation temperatures with ozonated POME, the maximum H₂ yield was 62 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 63 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹), respectively. The highest efficiency of total and soluble COD removal was obtained at 44 and 37%, respectively following the mesophilic fermentation, of 24 and 25%, respectively under the thermophilic fermentation, of 32 and 20%, respectively under the extreme-thermophilic fermentation. The production of volatile fatty acids increased with an increased fermentation time and temperature in both raw and ozonated POME under all three fermentation temperatures. The accumulation of volatile fatty acids in the reactor content were mostly acetic and butyric acids. H₂ fermentation under the mesophilic condition of 37 °C was the better selection than that of the thermophilic and extreme-thermophilic fermentation.     

Characteristics of the process of biohydrogen production from simple and complex substrates with different biopolymer composition

The work investigated the characteristics of the dark fermentation (DF) process of a number of simple (starch, sunflower oil, peptone, both separately and mixed) and complex (dog food, pig feed, sewage sludge) substrates using a mixed culture of microorganisms, with a controlled pH (5.5), at 55 °C. Peptone and sunflower oil were characterized by the lowest production of H₂, namely 5.0 and 2.3 ml H₂/g COD, respectively. The specific hydrogen yield from starch was 1.55 mol H₂/mol hexose. The addition of peptone and sunflower oil to starch reduced the specific yield of hydrogen from starch by 23%. A large difference in hydrogen production was observed during DF of complex substrates. The specific hydrogen yield from dog food was 46.5 ml H₂/g COD or 143.4 ml H₂/g carbohydrates; from pig feed – 32.1 ml H₂/g COD or 91.6 ml H₂/g carbohydrates; and from sewage sludge – 9.3 ml H₂/g COD or 98.0 ml H₂/g carbohydrates. Possible relationships between the biopolymer composition of substrates and characteristics of the DF process were analyzed using Spearman's rank correlation coefficients. The concentration of carbohydrates, as well as the ratio of carbohydrates/proteins and carbohydrates/fats, were the main factors influencing the high specific yield of H₂, its content in biogas, as well as the ratio of H₂/soluble metabolites. The concentration of proteins had a statistically significant positive effect on the accumulation of acetate and succinate, and carbohydrates - on the accumulation of caproate.     

Improving production of biohydrogen from COOH-functionalized multiwalled carbon nanotubes through Co-immobilization with Clostridium pasteurianum

Biohydrogen is a promising clean energy. Multiple researchers have successfully increased hydrogen production by augmenting nanomaterials with anaerobic hydrogen-producing microorganisms. Herein, a novel approach is developed in which a pure strain of Clostridium pasteurianum CH5 is co-immobilized with carboxylic-acid-functionalized multiwalled carbon nanotubes (MWCNT-COOH). The direct co-immobilization of C. pasteurianum CH5 with 800-mg/L MWCNT-COOH improves the hydrogen yield (HY) to 2.43 mol H2/mol glucose. When C. pasteurianum CH5 and 800 mg/L MWCNT-COOH are preincubated for 24 h, the HY increases to 2.38 mol H2/mol glucose, a 46.9% increase relative to the control (without co-immobilization) and a 133.3% increase relative to the conventional suspended growth of C. pasteurianum CH5 mixed with the same concentration of MWCNT-COOH. These results indicate that bringing nanomaterials into closer contact with microorganisms can serve as a feasible and simple approach for biohydrogen production.     

Comparative study on biohydrogen production by newly isolated Clostridium butyricum SP4 and Clostridium beijerinckii SP6

The hydrogen-producing bacteria SP4 and SP6 were isolated from the compost and identified by 16S rRNA gene sequencing as Clostridium butyricum and Clostridium beijerinckii, respectively. A comparative study on the biohydrogen-producing activity of the isolated strains was carried out using mono-, di- and tri-saccharides belonging to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose). To assess the biotechnological significance, real wastewater rich in sugars (cheese whey, confectionery wastewater, sugar beet processing wastewater) was also used as a substrate. C. butyricum SP4 fermented sugars with a yield of 0.93–1.52 mol H₂/mol hexose (pentose); the maximum yield was obtained from fructose, the minimum – from raffinose and cellobiose. The most preferred substrate for C. beijerinckii SP6 was sucrose with a yield of 1.76 mol H₂/mol hexose, while cellobiose yielded only 0.64 mol H₂/mol hexose. Overall, the efficiency of converting wastewater to H₂ by C. butyricum SP4 was also slightly lower (66–93 ml H₂/g chemical oxygen demand (COD)) than that of C. beijerinckii SP6 (76–103 ml H₂/g COD). Even though the main soluble metabolite products (SMPs) for both isolates were acetate and butyrate, C. butyricum SP4 also produced a significant amount of ethanol (up to 21.5% of SMPs) and formate (up to 32.5% of SMPs), and C. beijerinckii SP6 – lactate (up to 25% of SMPs). A distinctive feature of C. beijerinckii SP6 was a significantly lower (almost 2 times) yield of SMPs, while C. butyricum SP4 had a higher rate of H₂ production according to the results obtained from the kinetic study using the modified Gompertz equation and the first order equation. Analysis of Spearman's rank correlation coefficients revealed a statistically significant relationship between the kinetic parameters of H₂ production and the concentration of butyrate and the final pH of the medium for C. butyricum SP4, and with the concentration of ethanol for C. beijerinckii SP6. These findings provide valuable information on the metabolic capabilities of the most studied hydrogen-producing representatives of the Clostridium genus for their use in optimizing the technology for biohydrogen production by dark fermentation of various organic wastes.     

Techno-economic evaluation of biohydrogen production from wastewater and agricultural waste

The world is facing serious climate change caused in part by human consumption of fossil fuel. Therefore, developing a clean and environmentally friendly energy resource is necessary given the depletion of fossil fuels, the preservation of the earth's ecosystem and self-preservation of human life. Biological hydrogen production, using dark fermentation is being developed as a promising alternative and renewable energy source, using biomass feedstock. In this study, beverage wastewater and agricultural waste were examined as substrates for dark fermentation to produce clean biohydrogen energy.     

Synergistic strategy for the enhancement of biohydrogen production from molasses through coculture of Lactobacillus brevis and Clostridium saccharobutylicum

This study aimed to evaluate the capacity of different inoculum sources and their bacterial diversity to generate hydrogen (H₂). The highest Simpson (0.7901) and Shannon (1.581) diversity indexes for H₂-producing bacterial isolates were estimated for sewage inocula. The maximum cumulative H₂ production (H_max) was 639.6 ± 5.49 mL/L recorded for the sewage inoculum (SS30) after 72 h. The highest H₂-producing isolates were recovered from SS30 and identified as Clostridium saccharobutylicum MH206 and Lactobacillus brevis MH223. The H_max of C. saccharobutylicum, L. brevis, and synergistic coculture was 415.00 ± 24.68, 491.67 ± 15.90, and 617.67 ± 3.93 mL/L, respectively. The optimization process showed that the H_max (1571.66 ± 33.71 mL/L) with a production rate of 58.02 mL/L/h and lag phase of 19.33 h was achieved by the synergistic coculture grown on 3% molasses at 40 °C, pH 7, and an inoculum size of 25% (v/v). This study revealed the economic feasibility of the synergistic effects of coculture on waste management and biohydrogen production technology.     

Mathematical modelling for biohydrogen production by Clostridium beijerinckii

Hydrogen is an energy source that can be produced by Clostridium sporogenes microorganism. In the present work, modeling of dark fermentation using Clostridium beijerinckii and dextrose as substrate was performed to evaluate how the gases and liquid by-products affect the biological process. A mathematical model was developed according to ADM1. The developed model takes into account biochemical reactions, physicochemical equilibrium as well as mass transfer processes during dark fermentation. Findings revealed that Clostridium beijerinckii reached a yield as high as 3.58 mol of H₂/mol of dextrose and generates by-products in the aqueous phase that may either be used as raw materials in a chemical process. Clostridium beijerinckii is very sensitive to acid media (pH < 5.0) and shows a low rate of biohydrogen production (even the absence of metabolic activity) at pH lower than 4.5. The developed model is able to predict (R² > 0.95) dextrose consumption profile, cumulative biohydrogen production and the maximum concentrations of liquid by-products.