Escherichia coli wild type and hydrogenase mutant cells growth and hydrogen production upon xylose and glycerol co-fermentation in media with different buffer capacities

In this study, the new strategy for long term bio-hydrogen (H₂) production using different substrates and waste materials is presented. Growth characteristics and H₂ production were investigated upon consumption of 0.4% xylose and 1% glycerol alone (which were optimal) or their mixture by Escherichia coli BW25113 wild type parental strain (PS) and ΔhyaB, ΔhybC, ΔhycE, ΔhyfG mutants with genes deletions for key subunits of hydrogenase (Hyd)-1 to Hyd-4, respectively, in high and low buffer capacity peptone (HPM, LPM) mediums, pH 5.5 and 7.5. Overall, pH 5.5 negatively affected bacterial growth and H₂ production. At pH 7.5, apart from Hyd-3 and Hyd-4 mutants, upon growth of PS, Hyd-1 and Hyd-2 mutants drop of Pt redox electrode readings from positive (～+150 mV) to negative (of ?400 to ?550 mV) values was detected during log growth phase mentioning H₂ formation. Xylose and glycerol co-utilization did not affect PS and Hyd-1 and Hyd-2 mutant's biomass and H₂ formation during log growth phase in LPM, but ～1.5 fold stimulated these parameters, especially in HPM, pH 7.5, during prolonged 96 h bacterial growth. Roles of Hyd-3 and Hyd-4 in H₂ production; and Hyd-1 and Hyd-2 in H₂ oxidation during bacterial log growth phase were stated under xylose and glycerol co-fermenting conditions. The results obtained might be valuable for industrial long term H₂ production by bacteria using mixture of carbon sources and combining various organic waste materials.     

Enhanced hydrogen gas production from mixture of beer spent grains (BSG) and distiller's grains (DG) with glycerol by Escherichia coli

Escherichia coli perform mixed acid fermentation and produce hydrogen gas (H₂) as one of the fermentation end products. E. coli can ferment sugars like glucose, xylose and alcohols like glycerol. It has been shown that E. coli has the ability to utilize pretreated organic waste (BSG or DG) or mixtures of it with glycerol and H₂ can be produced. H₂ evolution was maximum when the concentration of BSG was 4% and DG - 10% yielding 1.4 mmol L⁻¹ H₂. H₂ evolution was prolonged to ~24–120 h when mixtures of glycerol and DG or BSG wastes were applied. Moreover, in hycE (lacking large subunit of Hyd-3) or hyfG (lacking large subunit of Hyd-4) single mutants H₂ production was absent compared to wild type suggesting that Hyd-3 and Hyd-4 are responsible for H₂ generation. In addition, multiple mutant enhanced cumulative H₂ production ~3–4 fold. Taken together it can be proposed that BSG or DG wastes either together or in mixture with glycerol can be applied to obtain E. coli biomass and produce bio-H₂. The novel data can be used to further control effectively the application of organic waste resources as a feedstock for developing bio-H₂ production technology.     

Hydrogen production by Escherichia coli during anaerobic utilization of mixture of lactose and glycerol: Enhanced rate and yield,prolonged production

Escherichia coli wild type has the ability to utilize lactose or the mixture of lactose and glycerol producing bio-hydrogen (H₂) at different pH values. At pH 7.5 in hyaB (lacking large subunit of hydrogenase (Hyd)-1) and hybC (lacking large subunit of Hyd-2) single mutants fermenting lactose (1 g L⁻¹) H₂ yield was ∼7- and 5-fold more, respectively, compared to the wild type. During the fermentation of lactose (1 g L⁻¹) and glycerol (10 g L⁻¹) mixture H₂ yield in wild type increased ∼3-fold, compared to fermenting lactose only. H₂ generation in wild type was monitored in batch cultures during 168 h of growth when utilizing the mixture of lactose and glycerol in all combinations of different concentration. In hyaB but not in hybC mutant H₂ evolution was detected till 240 h in the mixture of 5 g L⁻¹ lactose and 10 g L⁻¹ glycerol. The highest H₂ production rate of 21.94 mL L⁻¹ h⁻¹ was detected in hyaB mutant at pH 7.5 when 1 g L⁻¹ lactose was applied. The results showed optimized H₂ production using different mutants, lactose and its mixture with glycerol. They can be applied for renewable energy, especially bio-H₂ production.     

Thermophilic hydrogen and methane production from sugarcane stillage in two-stage anaerobic fluidized bed reactors

This study evaluated the feasibility of H₂ and CH₄ production in two-stage thermophilic (55 °C) anaerobic digestion of sugarcane stillage (5,000 to 10,000 mg COD.L⁻¹) using an acidogenic anaerobic fluidized bed reactor (AFBR-A) with a hydraulic retention time (HRT) of 4 h and a methanogenic AFBR (AFBR-S) with HRTs of 24 h–10 h. To compare two-stage digestion with single-stage digestion, a third methanogenic reactor (AFBR-M) with a HRT of 24 h was fed with increasing stillage concentrations (5,000 to 10,000 mg COD.L⁻¹). The AFBR-M produced a methane content of 68.4 ± 7.2%, a maximum yield of 0.30 ± 0.04 L CH₄.g COD⁻¹, a production rate of 3.78 ± 0.40 L CH₄.day⁻¹.L⁻¹ and a COD removal of 73.2 ± 5.0% at an organic loading rate (OLR) of 7.5 kg COD.m⁻³.day⁻¹. In contrast, the two-stage AFBR-A system produced a hydrogen content of 23.9 ± 5.6%, a production rate of 1.30 ± 0.16 L H₂.day⁻¹.L⁻¹ and a yield of 0.34 ± 0.08 mmol H₂.g CODap⁻¹. Additionally, the decrease in the HRT from 18 h to 10 h in the AFBR-S favored a higher methane production, improving the maximum methane content (74.5 ± 6.0%), production rate (5.57 ± 0.38 L CH₄.day⁻¹.L⁻¹) and yield (0.26 ± 0.06 L CH₄.g COD⁻¹) at an OLR of 21.6 kg COD.m⁻³.day⁻¹ (HRT of 10 h) with a total COD removal of 70.1 ± 7.1%. Under the applied COD of 10,000 mg L⁻¹, the two-stage system showed a 52.8% higher energy yield than the single-stage anaerobic digestion system. These results show that, relative to a single-stage system, two-stage anaerobic digestion systems produce more hydrogen and methane while achieving similar treatment efficiencies.     

Hydrogen production by Escherichia coli growing in different nutrient media with glycerol: Effects of formate,pH, production kinetics and hydrogenases involved

The Escherichia coli BW25113 or MC4100 wild type parental strains growth and H₂ production kinetics was studied in batch cultures of minimal salt medium (MSM) and peptone medium (PM) at pH of 5.5–7.5 upon glycerol (10 g L^?1) fermentation and formate (0.68 g L^?1) supplementation. The role of formate alone or with glycerol on growth and H₂ production via hydrogenases (Hyd) was investigated in double hyaB hybC (lacking large subunits of Hyd 1 and 2), triple hyaB hybC hycE (lacking large subunits of Hyds 1-3) and sole selC (lacking formate dehydrogenase H) mutants during 24 h bacterial growth. H₂ production was delayed and observed after 24 h bacterial wild type strains growth on MSM. Moreover, it reached the maximal values after 72 h growth at the pH 6.5 and pH 7.5. Biomass formation of the mutants used was inhibited ～3.5 fold compared with wild type, and H₂ production was absent in hyaB hybC hycE and selC mutants upon glycerol utilization on MSM at pHs of 5.5–7.5. Formate inhibited bacterial growth on MSM with glycerol, but enhanced and recovered H₂ production by hybC mutant at pH 7.5. H₂ evolution was delayed at pH 7.5 in PM, but observed and stimulated at pH 6.5 upon glycerol and formate utilization in hyaB hybC mutant. H₂ production was absent in hyaB hybC hycE and selC mutants upon glycerol, formate alone or with glycerol fermentation at pH 6.5 and pH 7.5; formate supplementation had no effect. The results point out E. coli ability to grow and utilize glycerol in MSM with comparably high H₂ yield: as well as they suggest the key role of Hyd-3 at both pH 6.5 and pH 7.5 and the role of Hyd-2 and Hyd-4 at pH 7.5 in H₂ production by E. coli during glycerol fermentation with formate supplementation. The results obtained are novel and might be useful in H₂ production biotechnology development using different nutrient media and glycerol and formate as feedstock.     

HRT control as a strategy to enhance continuous hydrogen production from sugarcane juice under mesophilic and thermophilic conditions in AFBRs

The objective of this study was to evaluate the effects of hydraulic retention time (HRT) (8–1 h) on H₂ production from sugarcane juice (5000 mg COD L⁻¹) in mesophilic (30 °C, AFBR-30) and thermophilic (55 °C, AFBR-55) anaerobic fluidized bed reactors (AFBRs). At HRTs of 8 and 1 h in AFBR-30, the H₂ production rates were 60 and 116 mL H₂ h⁻¹ L⁻¹, the hydrogen yields were 0.60 and 0.10 mol H₂ mol⁻¹ hexose, and the highest bacterial diversities were 2.47 and 2.34, respectively. In AFBR-55, the decrease in the HRT from 8 to 1 h increased the hydrogen production rate to 501 mL H₂ h⁻¹ L⁻¹ at the HRT of 1 h. The maximum hydrogen yield of 1.52 mol H₂ mol⁻¹ hexose was observed at the HRT of 2 h and was associated with the lowest bacterial diversity (0.92) and highest bacterial dominance (0.52).     

Ozonation aided mesophilic biohydrogen production from palm oil mill effluent

Ozone pretreatment of palm oil mill effluent (POME) was employed to improve sustrate biodegradability prior to biological H₂ production. The H₂ production was conducted at varing pHs from 4.0 to 6.0 to examine the impact of pH on the H₂ mesophilic production (37 °C). The optimal pH for H₂ production was 6.0 for both raw and ozonated POME. The POME concentrations were greatly influenced the yields and rates of H₂ production. At the optimal pH, the maximum H₂ production yield of 182 ± 7.2 mL.g⁻¹ COD (7.96 mmoL.g⁻¹ COD) was achieved at the ozonated POME concentration of 30,000 mg COD.L⁻¹. The maximum H₂ production rate (R_max) of 43.1 ± 2.5 mL.h⁻¹ was obtained at the ozonated POME concentration of 25,000 mg COD.L⁻¹. The highest total COD removal was 44% at of 15,000 mg COD.L⁻¹ ozonated POME. Acetic and butyric acids were dominant products during H₂ fermentation and tended to increase with the increased POME concentrations. Ozonation as a pretreatment process showed significant enhancement of the POME biodegradability , and subsequently improved the H₂ production H₂.     

Comparative evaluation of the mesophilic and thermophilic biohydrogen production at optimized conditions using tequila vinasses as substrate

The objective of this work was to comparatively evaluate the production of biohydrogen (bio-H₂) from tequila vinasses at optimized mesophilic and thermophilic conditions and to elucidate the main metabolic routes involved. Optimal temperatures of 35 °C and 55 °C, and pH of 5.5 maximized the bio-H₂ production rates, 25.5 ± 0.01 NmL h⁻¹ and 169.9 ± 8.9 NmL h⁻¹ in the mesophilic and thermophilic regimens, respectively. During the operation of anaerobic sequencing batch reactors, the thermophilic process allowed a volumetric bio-H₂ production rate of 519 ± 13 NmL-H₂ L⁻¹ d⁻¹ equivalent to 750 ± 19 NmL-H₂ L_vinasse⁻¹, while the mesophilic one 448 ± 23 NmL-H₂ L⁻¹ d⁻¹ and 647 ± 33 NmL-H₂ L_vinasse⁻¹, respectively. Furthermore, the gas produced under thermophilic conditions showed high hydrogen content (86.5%). Finally, formate degradation and glucose fermentation to acetic and butyric acids were the main metabolic routes involved in bio-H₂ production under thermophilic conditions, while at mesophilic conditions, the lactate and formate degradation pathways governed.     

Screening and optimization of trace elements supplement in sweet sorghum juice for ethanol production

Eight trace elements were screened for increasing efficiency of ethanol yield from sweet sorghum juice using the Plackett-Burman design method. MnCl₂·4H₂O, CoCl₂·6H₂O and biotin was screened as the significant variables which have positive effects on ethanol production from sweet sorghum juice. The values of MnCl₂·4H₂O, CoCl₂·6H₂O and biotin, optimized by Box-Behnken design method, were 7.70 mg L⁻¹, 15.74 mg L⁻¹ and 11.97 mg L⁻¹, respectively. The experimental efficiency of ethanol yield under optimal conditions was 89.30 ± 0.10%, which enhances the efficiency of ethanol yield by 5.63% by the addition of MnCl₂·4H₂O, CoCl₂·6H₂O and biotin. The results from this study have identified optimal conditions as a foundation for pilot scale ethanol production.     

Optimization of dilute acid and enzymatic hydrolysis for dark fermentative hydrogen production from the empty fruit bunch of oil palm

Pretreatment of the empty fruit brunch (EFB) from oil palm was investigated for H₂ fermentation. The EFB was hydrolyzed at various temperatures, H₂SO₄ concentrations, and reaction times. Subsequently, the acid-hydrolysate underwent enzymatic saccharification under various temperature, pH, and enzymatic loading conditions. Response surface methodology derived the optimum sugar concentration (SC), hydrogen production rate (HPR), and hydrogen yield (HY) as 28.30 g L⁻¹, 2601.24 mL H₂ L⁻¹d⁻¹, and 275.75 mL H₂ g⁻¹ total sugar (TS), respectively, at 120 °C, 60 min of reaction, and 6 vol% H₂SO₄, with the combined severity factor of 1.75. Enzymatic hydrolysis enhanced the SC, HY, and HPR to 34.52 g L⁻¹, 283.91 mL H₂ g⁻¹ TS, and 3266.86 mL H₂ L⁻¹d⁻¹, respectively, at 45 °C, pH 5.0, and 1.17 mg enzyme mL⁻¹. Dilute acid hydrolysis would be a viable pretreatment for biohydrogen production from EFB. Subsequent enzymatic hydrolysis can be performed if enhanced HPR is required.     

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.     

Inhibition by the ionic strength of hydrogen production from the organic fraction of municipal solid waste

Composition of the Organic Fraction of Municipal Solid Waste (OFMSW) in organic compounds and inorganic ions is highly variable and might impact the microbial activity in dark fermentation processes. In this study, the effect of the total amount of inorganic ions on fermentative hydrogen production was investigated. Batch experiments were carried out at pH 6 and under a temperature of 37 °C. A freshly reconstituted organic fraction of municipal solid waste (OFMSW) was used as model substrate. At low concentrations in ammonium or chloride ions (2.9–5.1 g L⁻¹, respectively), the hydrogen yield reached a maximum of 40.8 ± 0.5. mLH₂.gVS⁻¹ and 25.1 ± 5.6 mLH₂.gVS⁻¹. In contrast, at high total ionic concentrations of ammonium and chloride (11.1–35.5 g L⁻¹ respectively), a strong inhibition of the fermentative microbial activity and more particularly hydrogen production, was observed. When considering the ionic strength of each ion, the effects of ammonia, chloride or a mixture of different ions (Na⁺, K⁺, H⁺, Li⁺, NH₄⁺, Mn²⁺, NH₄⁺, Mg²⁺, Cl⁻, PO₄³⁻, Br⁻, I⁻, SO₄²⁻) showed very similar inhibitory trends regardless the type of ion or the composition of the ionic mixture. A threshold inhibitory value of the ionic strength was estimated at 0.75 ± 0.13 M with a substantial impact on the fermentative activity from 0.81 ± 0.12 M, with hydrogen yields of 18.1 ± 3.3 and 6.2 ± 4.1 mLH₂.gVS⁻¹, respectively. Microbial community composition was also significantly impacted with a specific decrease in relative abundance of hydrogen-producing bacteria from the genus Clostridium sp. at high ionic strength.     

Using a food and paper-cardboard waste blend as a novel feedstock for hydrogen production: Influence of key process parameters on microbial diversity

In this study, fermentation of a thermally treated simulated organic solid waste into hydrogen (H₂) was examined using a pretreated anaerobic mixed culture. The culture was fed a steam exploded food waste plus paper-cardboard waste blend liquor with and without linoleic acid (LA). The individual and interaction effects of the initial pH, LA concentration and the initial chemical oxygen demand (COD) concentration on H₂ and methane (CH₄) production was assessed using a Box–Behnken design (BBD). The BBD model predicted a maximum H₂ yield of 87 mL g⁻¹ COD or 98 mL H₂ g⁻¹ VS with 1.6 g L⁻¹ LA, an initial pH of 5.93 and an initial COD of 9.34 g COD L⁻¹. The major microbial populations detected in cultures at pH 5.5 with and without LA included Clostridium sp., Enterococcus asini, Enterococcus faecalis, and Lactobacillus gallinarum. The dendrogram for the 16S rRNA gene T-RFs profiles showed four major groups with a similarity index of 72–75% for Clade III. The major H₂-producing populations were grouped in Clade I with a similarity index range of 55–75%.     

Evaluation of feeding strategies in upflow anaerobic sludge bed reactor for hydrogenogenesis at psychrophilic temperature

The present work evaluated the biohydrogen production from a 0.4 L upflow anaerobic sludge blanket reactor type (UASB) operating at psychrophilic temperature (21 ± 2 °C) at different feeding strategies varying hydraulic retention times (HRT) and sucrose concentration in the feeding. First strategy (24 h/31c) fed semi-continuously 31 g_sucrose L⁻¹ at 24 h HRT; second strategy (12 h/19c) fed semi-continuously 19 g_sucrose L⁻¹ at 12 h HRT; third strategy (4 h/8c) fed continuously 8.3 g_sucrose L⁻¹ at 4 h HRT.After 70 days of operation, the UASB accumulated 65.44 L H₂. The average HY for the whole operation during the three strategies was 62.6 NmL H₂ g_sucrose⁻¹, and average hydrogen content was 69.04%. In general terms, the best operation strategy was 12 h/19c since it presented good set of results, the best HY (70.6 NmL H₂ g_sucrose⁻¹) and a comparable hydrogen production rate (2.6 L (L d)⁻¹) to that obtained in 4 h/8c strategy (3.17 L (L d)⁻¹). The average gross energy potential rate from the 12 h/19c strategy was 46.21 kJ (L d)⁻¹, whereas energy heating losses were circumvented due to operation at psychrophilic regime. Indeed, psychrophilic or room temperatures should be broadly regarded as an effective alternative towards net energy gains in biohydrogen production.     

Improvement of biohydrogen production from brewery wastewater: Evaluation of inocula,support and reactor

This work explores the production of biohydrogen from brewery wastewater using as inoculum a culture produced by natural fermentation of synthetic wastewater and Klebsiella pneumoniae isolated from the environment. Klebsiella pneumoniae showed good performance as inoculum, as evaluated using assays of between 9 and 16 cycles, with durations of 12 and 24 h, carbohydrate concentrations from 2.79 to 7.22 g L⁻¹, and applied volumetric organic loads from 2.6 to 12.6 g carbohydrate L⁻¹ day⁻¹. The best results were achieved with applied volumetric organic loads of 12.6 g carbohydrate L⁻¹ day⁻¹ and cycle length of 12 h, resulting in mean volumetric productivity of 0.88 L H₂ L⁻¹ day⁻¹, maximum molar flow of 10.80 mmol H₂ h⁻¹, and mean yield of 0.70 mol H₂ mol⁻¹ glucose consumed. The biogas H₂ content was between 18 and 42%, while the mean organic compounds removal and carbohydrate conversion efficiencies were 23 and 81%, respectively. The inoculum produced by natural fermentation was not viable.     

Thermophilic biohydrogen recovery from palm oil mill effluent

Enhancement of biological H₂ production efficiency with pre-ozonation process of palm oil mill effluent (POME) prior to thermophilic dark fermentation (55 °C) was investigated. H₂ fermentation experiments were conducted using varying concentrations of raw and ozonated POME. Results revealed that H₂ can be produced from both raw and ozonated POME under thermophilic fermentation. Maximum H₂ production yield of 77 mL.g⁻¹COD_removed was obtained from ozonated POME, which was higher than that of 51 mL·g⁻¹ COD_removed obtained from raw POME at the highest concentration of 35,000 mg COD.L⁻¹. Meanwhile, the specific H₂ production rate (R'_max) of 1.9 and 1.5 mL·h⁻¹·g⁻¹ TVS were observed in raw and ozonated POME at the concentration of 25,000 mg COD.L⁻¹, respectively. The main metabolic products during POME fermentation were acetic and butyric acids and trace amount of valeric acid. Propionic acid and ethanol have contributed, which could be reduced H₂ production in all batch experiments for both POME. The highest efficiency of total and soluble COD removal of 24 and 25% was obtained from the raw POME, and those of 19 and 25% was obtained from the ozonated POME. The present study demonstrates that the POME loading was greatly influenced on the H₂ production yields and rates. The comparative results showed that the ozonated POME gave higher H₂ yields than the raw POME. Thus, demonstrating that the ozonation process significantly improved the POME biodegradability, which is able to enhance H₂ production yields. However, the ozone pre-treatment was not improved in the specific H₂ production rates.     

Quantitative real-time PCR monitoring dynamics of Thermotoga neapolitana in synthetic co-culture for biohydrogen production

This study demonstrates the potential for biohydrogen production in a co-culture of two ecologically distant species, Thermatoga neapolitana and Caldicellulosiruptor saccharolyticus, and the development of a quantitative real-time PCR (qPCR) method for quantifying the hyperthermophilic bacterium of the genus Thermotoga. Substrate utilization and H₂ production performance was compared to those of their individual cultures. The highest H₂ yields obtained were 2.7 ± 0.05, 2.5 ± 0.07 and 2.8 ± 0.09 mol H₂/mol glucose for C. saccharolyticus, T. neapolitana, and their co-culture respectively. Statistical analysis comparing the H₂ production rate of the co-culture to either C. saccahrolyticus or T. neapolitana pure cultures indicated a significant difference in the H₂ production rate (p < 0.05: t-test), with the highest rate of H₂ production (36.02 mL L⁻¹ h⁻¹) observed from the co-culture fermentations. In order to monitor the presence of T. neapolitana in the bioprocess, we developed a qPCR method using 16S rRNA gene and hydrogenase (hydA) gene targets. The qPCR data using hydA primers specific to T. neapolitana showed an increase in hydA gene copies from 3.32 × 10⁷ to 4.4 × 10⁸ hydA gene copies per mL confirming the influence of T. neapolitana in the synthetic consortium.     

Extreme thermophilic condition: An alternative for long-term biohydrogen production from sugarcane vinasse

Boosted by the high temperatures in which vinasse is generated (90 °C–100 °C), this study evaluated the effect of an extreme thermophilic condition (70 °C) on sugarcane vinasse Dark Fermentation (DF) in an Anaerobic Structured Bed Reactor (ASTBR). Four hydraulic retention times (HRT) (19, 15, 12 and 8 h) were evaluated. Higher HRT resulted in a greater H₂ production rate (690 mLH₂.d⁻¹.L⁻¹), higher yields (1.8 molH₂.mol_Glucose⁻¹) and greater stability. The extreme temperature inhibits microorganisms' extracellular polymer production, thus leading to a disperse growth, preventing excess biomass accumulation, which was previously reported as the main drawback in H₂ production at lower temperatures. The ASTBR higher void index is also responsible for lower biomass/solids retention. The H₂ production main route was through the lactic/acetic acid pathway, which is highly reliant on the pH of fermentation broth. The main genus involved in H₂ production at 70 °C were Clostridium, Pectinatus, Megasphaera and Lactobacillus.     

Energy valorization of crude glycerol and sanitary sewage in hydrogen generation by biological processes

Biohydrogen production was studied with increased concentrations of crude glycerol (CG) co-digested with sanitary sewage by anaerobic consortium bacteria in anaerobic batch reactors, at 30 °C and initial pH 7.0. The CG was obtained during the biodiesel production from waste cooking oils (WCO). The anaerobic consortium was from a granular sludge of UASB reactor used in the treatment of poultry wastes heat treated to inhibit methanogenic activity previously. The higher H₂ generation was observed with 240.0 g COD L⁻¹ (35.82 mmol L⁻¹), being consumed 63.9% of CG. The co-digestion associated with sanitary sewage facilitated the CG consumption with the increase of organic load, favoring the acetic acid and 1,3-propanediol production in a liquid phase of the reactors. These results are promising, contributing effectively to the treatment of both wastes with concomitant generation of bioenergy.     

Defining the roles of the hydrogenase 3 and 4 subunits in hydrogen production during glucose fermentation: A new model of a H2-producing hydrogenase complex

Hydrogen (H₂)-producing hydrogenase (Hyd) activity of E. coli wild type and mutants with defects in subunits of Hyd-3 or Hyd-4 during fermentation at different glucose concentrations and pHs was studied. Hyd-3 was mainly responsible for H₂ production but a significant contribution by Hyd-4 to total H₂ production depended on the glucose concentration and pH. Surprisingly, not all Hyd-3 or Hyd-4 subunits contributed towards H₂ production. Hyd-4 mainly exhibited H₂-oxidizing activity in cells growing on 0.2% glucose at pH 7.5, while at pH 5.5 it had a significant impact on H₂ production. Importantly, a hyfG mutant (lacking the large subunit of Hyd-4) had a ~2.2 fold decrease in H₂ production when cells were grown with 0.2% glucose. A similar role of Hyd-4 was shown at pH 6.5 grown with 0.8% glucose. This study provides new information to allow improvements in H₂ production yield and in our general understanding of H₂ metabolism.