Ferric oxide/date seed activated carbon nanocomposites mediated dark fermentation of date fruit wastes for enriched biohydrogen production

Biohydrogen production from biomass waste, not only addresses the energy demand in a renewable manner but also resolves the safe disposal issues associated with these biowastes. Also, scalable and low-cost techniques to enhance biohydrogen production have gained more attraction and are highly explored. In this research work, date-palm fruit wastes have been studied for their biohydrogen production potential using Enterobacter aerogenes by dark fermentation. Hydrogen yield and productivity were improved through the addition of iron oxide nanoparticles (Fe₃O₄ NPs) and its date seed activated carbon nanocomposites (Fe₃O₄/DSAC) to the fermentation media. Studies on discrete inclusions of these NPs showed that the appropriate dosage of NPs promoted, while higher dosages repressed the hydrogen production performance. Optimal dosage and fermentation time was observed as 150 mg/L and 24 h for both the additives. Fe₃O₄/DSAC nanocomposites showed better hydrogen production enhancement than Fe₃O₄ NPs. Maximum hydrogen yield of 238.7 mL/g was obtained for the 150 mg/L nanocomposites, which was 65.7% higher than that of the standalone Fe₃O₄ NPs and three folds higher than the yield of the control run without any NPs inclusion (78.4 mL/g). Metabolites analysis showed that the hydrogen evolution followed the ethanol-acetate pathway. Formation levels of longer chain propionate and butyrate co-metabolites were significantly low in the presence of Fe₃O₄/DSAC than Fe₃O₄. The carbon support in the nanocomposites acted as an adsorbent-buffer, which favored the medium pH in-addition to the stimulatory effects of Fe₃O₄ NPs. Cell growth and specific hydrogenase activity analysis were also performed to supplement the hydrogen production results. Gompertz and modified Logistic kinetic models were employed for kinetic modeling of experimental hydrogen production values. The Fe₃O₄/DSAC nanocomposites exhibited significant application potential for the production of biohydrogen from date fruit wastes.     

Biohydrogen production via integrated sequential fermentation using magnetite nanoparticles treated crude enzyme to hydrolyze sugarcane bagasse

This study presents a potential approach to enhance integrated sequential biohydrogen production from waste biomass using magnetite nanoparticle (Fe₃O₄ NPs) which is synthesized through waste seeds of Syzygium cumini. Consequences of 0.5% Fe₃O₄ NPs have been investigated on the thermal and pH stability of fungal crude cellulase. It is noticed that Fe₃O₄ NPs treated enzyme and control exhibits 100% activity in the temperature range of 45–60 °C and 45–55 °C, respectively. Moreover, Fe₃O₄ NPs treated enzyme showed extended thermal stability in the temperature range of 50–60 °C up to 12 h. Beside this, Fe₃O₄ NPs treated enzyme possesses 100% stability in the pH range of 5.0–7.0 whereas control exhibited only at pH 6.0. Enzymatic hydrolysis via Fe₃O₄ NPs treated enzyme has been employed which produces ～68.0 g/L reducing sugars from sugarcane bagasse. Subsequently, sugar hydrolyzate has been utilized as substrate in the sequential integrated fermentation that produces ～3427.0 mL/L cumulative hydrogen after 408 h. This approach may have potential for the pilot scale production of biohydrogen from waste biomass at low-cost in an eco-friendly manner.     

Comparison of magnetite/reduced graphene oxide nanocomposites and magnetite nanoparticles on enhancing hydrogen production in dark fermentation

Magnetite/reduced graphene oxide nanocomposites (Fe₃O₄-rGO NCs) and magnetite nanoparticles (Fe₃O₄ NPs) were added to enhance biohydrogen (bioH₂) production in dark fermentation. Concentration of supplements from 10 to 100 mg/L was appropriate to enhance bioH₂ production, and inhibition appeared once concentration exceeded 100 mg/L. The best bioH₂ yield was 198.30 mL/g glucose at 100 mg/L Fe₃O₄ NPs and 225.60 mL/g glucose at 100 mg/L Fe₃O₄-rGO NCs, which was 42.97% and 62.65% higher than that in the blank group, respectively. Both Fe₃O₄ NPs and Fe₃O₄-rGO NCs could intensify butyrate-type fermentation and change the hydrogen-producing microorganism cells morphology, but the enhancement effect of Fe₃O₄-rGO NCs was superior. Microbial community structure analysis showed that Clostridium-sensu-stricto-1 became more dominant ultimately by Fe₃O₄-rGO NCs.     

Novel cake-like Fe–N–C hybrid for H2 activation

The activation and utilization of hydrogen energy is an effective method to solve the global energy crisis and environmental pollution. Herein, biomass-derived Fe–N–C catalysts for H₂ activation were synthesized via the imitation of sponge cake baking. The sample pyrolyzed at 500 °C (Fe–N–C-500) presented the well-defined cake-like architecture with uniform distribution of Fe₃O₄ nanoparticles (NPs). Doping N species dispersed around metallic NPs in high density. Fe–N–C-500 exhibited excellent performance in the catalytic hydrogenation of nitrobenzene. The activity of Fe–N–C-500 depended on Fe₃O₄ NPs and pyridinic N, rather than Fe–N. The typical core-shell structure deemed vital for H₂ activation in previous reports was not necessary. Notably, water could significantly promote the H₂ activation, which might establish the communication between hydrogen molecules adsorbed on Fe₃O₄ NPs and doping N species through hydrogen bonds. Moreover, low temperature pyrolytic Fe–N–C-500 exhibited excellent stability and provided a promising potential for selective hydrogenation of nitroarenes or alkyne by regulating the reaction condition. This work provides an innovative approach to construct heterogeneous catalysts for H₂ activation.     

Effects of Fe0 and Ni0 nanoparticles on hydrogen production from cotton stalk hydrolysate using Klebsiella sp. WL1316: Evaluation of size and concentration of the nanoparticles

Fe⁰ and Ni⁰ nanoparticles (NPs) of certain size were synthesized and added to the hydrogen production system from cotton stalk hydrolysate using Klebsiella sp. WL1316. Fe⁰ and Ni⁰ NPs with a size of 50 nm at all concentrations effectively improve hydrogen production during mid to late fermentation stages; particularly, the highest daily hydrogen production obtained following treatment with 50 nm Fe⁰ NPs at 30 mg/L fermented for 96 h significantly increased by 61% comparing to the control treatment. The reducing sugar consumption in cotton stalk hydrolysate and ΔOD₆₀₀ could be improved to some extent by Fe⁰ and Ni⁰ NPs supplementation. Addition of Fe⁰ or Ni⁰ NPs of 50 nm at a concentration of 30 mg/L resulted in enhanced cumulative hydrogen production with improvement of hydrogen yield reached higher than 20%, and the values of Y(H₂/S) were all higher than 90 mL/g _substrate, reflecting good hydrogen production and substrate consumption. The analysis of the main soluble metabolites profile revealed that supplementation with Fe⁰ and Ni⁰ NPs of suitable size and concentration may decrease the metabolic flux in the competitive branch of hydrogen production and increase the metabolic flux of the key node that leads to hydrogen generation, thus promoting biohydrogen synthesis.     

Simultaneous thermophilic hydrogen production and phenol removal from palm oil mill effluent by Thermoanaerobacterium-rich sludge

Thermoanaerobacterium-rich sludge was used for hydrogen production and phenol removal from palm oil mill effluent (POME) in the presence of phenol concentration of 100–1000 mg/L. Thermoanaerobacterium-rich sludge yielded the most hydrogen of 4.2 L H₂/L-POME with 65% phenol removal efficiency at 400 mg/L phenol. Butyric acid and acetic acid were the main metabolites. The effects of oil palm ash, NH₄NO₃ and iron concentration (Fe²⁺) on hydrogen production and phenol removal efficiency from POME by Thermoanaerobacterium-rich sludge was investigated using response surface methodology (RSM). The RSM results indicated that the presence of 0.2 g Fe²⁺/L, 0.3 g/L NH₄NO₃ and 20 g/L oil palm ash in POME could improved phenol removal efficiency, with predicted hydrogen production and phenol removal efficiency of 3.45 L H₂/L-POME and 93%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with hydrogen production and phenol removal efficiency of 3.43 ± 0.12 L H₂/L-POME and 92 ± 1.5%, respectively. Simultaneous hydrogen production and phenol removal efficiency in continuous stirred tank reactor at hydraulic retention time (HRT) of 1 and 2 days were 4.0 L H₂/L-POME with 85% and 4.2 L H₂/L-POME with 92%, respectively. Phenol degrading Thermoanaerobacterium-rich sludge comprised of Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium aciditolerans, Desulfotomaculum sp., Bacillus coagulans and Clostridium uzonii. Phenol degrading Thermoanaerobacterium-rich sludge has great potential to harvest hydrogen from phenol-containing wastewater.     

Isolation of bacteria capable of hydrogen production in dark fermentation and intensification of anaerobic granular sludge activity

In the anaerobic biological treatment of pulp and papermaking wastewater, the gradual deposition of CaCO₃ eventually leads to the inhibition of the activity of anaerobic granular sludge. In this study, a hydrogen production bacterial Raoultella DW01 was isolated from domesticated anaerobic granular sludge. The fermentation conditions were designed using central composite design, and the optimum conditions obtained by response surface analysis encompassed an initial pH 5.77, 4.13 g/L l-glutamic acid and an inoculation amount of 15%. The H₂ production yield represented a 29.5% increase over the unoptimized conditions. Finally, the effect of adding DW01 on the biogas production in anaerobic granular sludge with different sludge ages was investigated. The cumulative biogas yield and the max biogas production rate increased by 27.8% and 53.5% after adding DW01 to a sludge with an age of 335 days compared with the on-intensified sludge. This paper provides a way to alleviate the CaCO₃ deposition by intensifying the activity of H₂ and acid-producing bacteria via improving the activity of granular sludge.     

Innovative hydrogen release from sodium borohydride hydrolysis using biocatalyst-like Fe2O3 nanoparticles impregnated on Bacillus simplex bacteria

For the first time in this innovative study, microorganisms such as Bacillus simplex bacteria, mostly used in biological activity studies, are used as a bio-supporter agent of iron to release hydrogen from sodium borohydride hydrolysis at 25.0 ± 0.1 °C. The goal is to investigate thoroughly sodium borohydride hydrolysis catalyzed by Fe₂O₃ nanoparticles impregnated on microorganism such as Bacillus simplex (BS) bacteria (Fe₂O₃@BS NPs) known with strong antibacterial properties, which makes innovative them a candidate for hydrolysis reaction. This study was focused on the preparation, identification, and catalytic use of biocatalyst-like Fe₂O₃@BS NPs for hydrogen release from the sodium borohydride hydrolysis at 25.0 ± 0.1 °C. The characterization results made after and before hydrolysis reaction using by SEM/SEM-EDX, FT-IR, XRD, UV–vis, XPS, DLS, ELS Zeta potential, ESR, and TEM techniques reveal the formation of highly active, stable, durable, and long-lived biocatalysts-like Fe₂O₃@BS NPs.     

Hydrogen production from sludge of poultry slaughterhouse wastewater treatment plant pretreated with microwave

This study aims to produce hydrogen from sludge of poultry slaughterhouse wastewater treatment plant (5% total solid) by anaerobic batch fermentation with Enterobactor aerogenes or mixed cultures from hot spring sediment as the inoculums. Sludge was heated in microwave at 850 W for 3 min. Results indicated that a soluble chemical oxygen demand (sCOD) of pretreated sludge was higher than that of raw sludge. Pretreated sludge inoculated with E. aerogenes and supplemented with the Endo nutrient had a higher hydrogen yield (12.77 mL H₂/g tCOD) than the raw sludge (0.18 mL H₂/g tCOD). When considered the hydrogen yield, the optimum initial pH for hydrogen production from microwave pretreated sludge was 5.5 giving the maximum value of 12.77 mL H₂/g tCOD. However, when considered the hydrogen production rate (R_m), the optimum pH for hydrogen production would be 9.0 with the maximum R_m of 22.80 mL H₂/L sludge·h.     

Enhanced biohydrogen production from corn stover hydrolyzate by pretreatment of two typical seed sludges

Five individual pretreatment methods (heat, ultrasonic, ultraviolet, acid, and base) were performed on two typical seed sludges (river sediments and anaerobic granular sludge) to evaluate their effectiveness on enriching efficient hydrogen (H₂)-producing bacteria and enhancing H₂ production using corn stover hydrolyzate. Results indicated that pretreatment processes caused more remarkable improvements for river sediments than anaerobic granular sludge. Among the five protocols, heat pretreatment reached high H₂ yield for both river sediments (4.17 mmol H₂/g utilized sugar) and anaerobic granular sludge (2.84 mmol H₂/g utilized sugar). Ultraviolet and ultrasonic pretreatments were conditionally effective for river sediments and anaerobic granular sludge, respectively. In most cases, pretreatment processes altered soluble metabolites distribution towards more acetate and less ethanol production. Microbial community analysis indicated that heat and ultrasonic pretreatments can respectively lead to significant and indistinctive change on original microbial community. Besides frequently detected Escherichia spp., Serratia spp., and Klebsiella spp., some species of Clostridium spp. and Bacillus spp. might be efficient H₂ producer responsible for better H₂-producing performances.     

Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation

Hydrogen formation performances of different anaerobic bacteria were investigated in batch dark fermentation of waste wheat powder solution (WPS). Serum bottles containing wheat powder were inoculated with pure cultures of Clostridium acetobutylicum (CAB), Clostridium butyricum (CB), Enterobacter aerogenes (EA), heat-treated anaerobic sludge (ANS) and a mixture of those cultures (MIX). Cumulative hydrogen formation (CHF), hydrogen yield (HY) and specific hydrogen production rate (SHPR) were determined for every culture. The heat-treated anaerobic sludge was found to be the most effective culture with a cumulative hydrogen formation of 560 ml, hydrogen yield of 223 ml H₂ g⁻¹ starch and a specific hydrogen production rate of 32.1 ml H₂ g⁻¹ h⁻¹.     

Hydrogen production by mixed bacteria through dark and photo fermentation

Mixed bacteria were used to improve hydrogen yield from cassava starch in combination of dark and photo fermentation. In dark fermentation, mixed anaerobic bacteria (mainly Clostridium species) were used to produce hydrogen from cassava starch. Substrate concentration, fermentation temperature and pH were optimized as 10.4 g/l, 31 °C and 6.3 by response surface methodology (RSM). The maximum hydrogen yield and production rate in dark fermentation were 351 ml H₂/g starch (2.53 mol H₂/mol hexose) and 334.8 ml H₂/l/h, respectively. In photo fermentation, immobilized mixed photosynthetic bacteria (PSB, mainly Rhodopseudomonaspalustris species) were used to produce hydrogen from soluble metabolite products (SMP, mainly acetate and butyrate) of dark fermentation. The maximum hydrogen yield in photo fermentation was 489 ml H₂/g starch (3.54 mol H₂/mol hexose). The total hydrogen yield was significantly increased from 402 to 840 ml H₂/g starch (from 2.91 to 6.07 mol H₂/mol hexose) by mixed bacteria and cell immobilization in combination of dark and photo fermentation.     

Enhanced anaerobic treatment of azo dye wastewater via direct interspecies electron transfer with Fe3O4/sludge carbon

In this study, enhancement of anaerobic treatment of azo dye wastewater with Fe₃O₄/SC is investigated. Fe₃O₄/sludge carbon (SC) is prepared from waste activated sludge using a simple impregnation method. Two identical laboratory-scale upflow anaerobic sludge blanket reactors with a working volume of 1.5 L are filled with 2 g/L of SC (reactor 1) and 2 g/L of Fe₃O₄/SC (reactor 2). Another reactor (reactor 0) is operated as a control. FTIR, XPS, SEM, and BET results indicate that Fe₃O₄ is successfully loaded on the SC. Addition of Fe₃O₄/SC to the reactor results in high chemical oxygen demand (78.13%) and color (97.60%) removal rates. Furthermore, Fe₃O₄/SC effectively decreases the amount of aniline generated and increases CH₄ production. The Fe₃O₄/SC anaerobic system is stable with fast recovery in terms of both organic matter removal and aniline control with high toxic substance concentrations and fluctuating organic loads. Moreover, Fe₃O₄/SC greatly enhances enzymatic activity and sludge granulation, which could greatly improve anaerobic reactor stability. High electron transport system and conductivity values and the enrichment of Geobacter and Methanosaeta species confirm that direct interspecies electron transfer occurs with Fe₃O₄/SC in the reactor. These results provide a scientific basis for further applications to azo dye wastewater treatment and promote resource utilization of waste sewage sludge.     

Bioconversion of crude glycerol from waste cooking oils into hydrogen by sub-tropical mixed and pure cultures

This study compared the biohydrogen generation by sub-tropical mixed and pure cultures from the crude glycerol from the biodiesel production using waste cooking oils (WCO). The crude glycerol was pretreated by pH adjustment. The mixed culture was obtained from a subtropical granular sludge of the UASB (Upflow Anaerobic Sludge Blanket) reactor used in the treatment of vinasse from sugarcane of ethanol and sugar industry. It was heat treated in order to inactivate hydrogen-consuming bacteria, which was identified by Illumina MiSeq Sequencing with a relative abundance of 97.96% Firmicutes Philum, 91.81% Clostridia Class and 91.81% Clostridiales Order. The pure culture was isolated from a sub-tropical granular sludge from UASB reactor of treating brewery wastewater and identified as Enterobacter sp. (KP893397). Two assays were carried in anaerobic batch reactors in order to verify the hydrogen production from crude glycerol bioconversion with: (I) mixed culture and (II) pure culture. The experiments were conducted at 37 °C, initial pH of 5.5 for assay I and 7.0 for assay II, with 20 g COD L⁻¹ of crude glycerol. The crude glycerol consumption was 56.2% and 88.0% for the assay I and II, respectively. The hydrogen yields were 0.80 moL H₂ mol⁻¹ glycerol for the assay I and 0.13 moL H₂ mol⁻¹ glycerol for the assay II. Enterobacter sp. preferred the reductive metabolic route, generating 1460.0 mg L⁻¹ of 1,3-propanediol, and it showed to be more sensitive in the presence of methanol from crude glycerol than mixed culture that preferred the oxidative metabolic route with biohydrogen generation. The mixed culture was more able to generate H₂ than pure culture from the crude glycerol coming from the biodiesel production using WCO.     

Co-fermentation of sewage sludge and algae and Fe2+ addition for enhancing hydrogen production

Co-fermentation of sewage sludge and algae was performed for enhancing the hydrogen production, and the effect of Fe²⁺ on co-fermentation process was examined. Results showed that both co-fermentation process and Fe²⁺ addition promoted hydrogen production. Highest hydrogen production of 28 mL/100 mL (14.8 mL H₂/g VS_added) was obtained from the co-fermentation group with 600 mg/L Fe²⁺ addition, which was 2.15 times, 2.00 times and 1.87 times of mono-fermentation of sludge, mono-fermentation of algae, and the co-fermentation group without Fe²⁺ addition. Both volatile solids and protein degradation were stimulated by co-fermentation process. Microbial analysis showed that co-fermentation groups with Fe²⁺ addition enriched Clostridium sensu stricto 13, Clostridium tertium and Terrisporobacter, which were positively correlated with cumulative hydrogen production. This study suggested that the co-fermentation of sludge and algae in the presence of Fe²⁺ could significantly improve the hydrogen production by stimulating the hydrogen-producing metabolism.     

Bioaugmentation on hydrogen production from food waste

The aim of this work was to evaluate the effect of two hydrolytic (Paenibacillus polymyxa and Bacillus subtilis) and two fermentative (Clostridium saccharobutylicum and Clostridium beijerinckii) strains on hydrogen (H₂) production in dark fermentation by batch testing. Food waste was used as a substrate, pretreated anaerobic sludge was used as the inoculum, and different concentrations of the evaluated microorganisms were used. Bioaugmentation with 3.5 × 10⁹ CFU/mL/L_reactor B. subtilis showed the best performance, obtaining a production of 84.5 mL H₂/g SV and a reduction in the lag phase (from 7.9 h in control to 3.5 h). Bioaugmentation with B. subtilis in an anaerobic sequencing batch reactor exhibited a significant effect on volumetric productivity, reaching a maximal increase of 344% of H₂ production in comparison with that obtained without the addition of the strain. The increase in H₂ was observed in a short period of time (4 cycles), after which H₂ production returned to the original H₂ production baseline. During all reactor operations, the main volatile fatty acids produced were acetic acid and butyric acid. Microbial community analysis when bioaugmentation was applied showed an importance of lactic acid bacteria abundance, such as that of Bifidobacterium and Lactobacillus, whose metabolic activity was crucial in reactor performance. The added concentration of microorganisms is a critical parameter for the bioaugmentation process.     

CeO2 modified Fe2O3 for the chemical hydrogen storage and production via cyclic water splitting

The chemical hydrogen storage (hydrogen reduction) and production (water splitting) behaviour of Ce-modified Fe₂O₃ mixed oxides were investigated. Fe_1−xCe_xO_2−δ (x = 0, 0.05, 0.1, 0.2, 0.3, 0.4 and 1) oxides prepared by chemical precipitation were characterized by XRD (X-ray diffraction), H₂-TPR (hydrogen temperature-programmed reduction) and H₂O-TPO (steam temperature-programmed oxidation) tests. XRD results showed that two kinds of Fe–Ce–O solid solutions (Ce-based and Fe-based) coexisted in Fe–Ce mixed oxides. H₂-TPR experiment suggested that Ce addition could reduce hydrogen reduction temperature while H₂O-TPO experiments over reduced oxides showed that Fe–Ce mixed oxides could split water to produce hydrogen at a lower temperature and complete in a shorter time. Both redox reactions (hydrogen reduction and water splitting) were sensitive to the temperature and active at a high temperature. The successive redox cycles were carried out over the Fe_0.7Ce_0.3O_2−δ mixed oxide at 750 °C. It kept a stable production of hydrogen in the successive redox process at the condition of serious agglomeration of the materials. The highest hydrogen storage amount was up to 1.51 wt% for the Fe–Ce sample with a 30% substitution of Ce for Fe.     

Hydrogen production in reactors: The influence of organic loading rate,inoculum and support material

Hydrogen production was evaluated in two thermophilic structured bed (USBR) reactors. USBR1was inoculated with auto-fermented sugarcane vinasse and low-density polyethylene cubes were used as support material. USBR2 was inoculated with anaerobic sludge from an up-flow anaerobic sludge blanket (UASB) reactor treating sugarcane vinasse, and polyurethane foam matrices was used as support material. The reactors were operated in parallel with sugar cane molasses at organic loading rate (OLR) from 30 to 120 g COD L⁻¹d⁻¹ during 45 days. Hydrogen production was detected during the whole operational period, with maximum values of 1123 mL H₂ d⁻¹L⁻¹ and 2041 mL H₂ d⁻¹L⁻¹ for USBR1 and USBR2, respectively. The number of gene copies encoding for Fe-hydrogenase was higher in USBR2 for all OLR applied. DNA sequences related to Thermoanaerobacterium and Clostridium sensu stricto were predominant in USBR1. In USBR2, in addition to these microorganisms, Lactobacillus, Pseudomonas and Thermotuga, and sequences with low frequency of abundance (<5%) involved directly and indirectly in hydrogen production were also present. The taxonomical and functional more diverse inoculum of USBR2 was associated with a higher hydrogen production. Besides fermentation, an unknown metabolism was relevant in USBR2, revealing the importance of physiological characterization of the microbial community present.     

Comparison of thermophilic and hyperthermophilic dark fermentation with subsequent mesophilic methanogenesis in expanded granular sludge bed reactors

Herein, dark fermentation (DF, V = 5.5 L) and subsequent mesophilic methanogenesis (V = 43.5 L) are run as expanded granular sludge bed reactors (EGSB) at thermophilic (υDF = 60 °C) and hyperthermophilic (υDF = 80 °C) temperatures. A synthetic glucose wastewater is run with a 22.5 g/L chemical oxygen demand (COD) and 48–9 h hydraulic retention times (HRTs), giving organic loading rates (OLRs) of 11–60 g COD/L/d for DF. The maximum hydrogen production rate (HPR) is HPR = 3.0 m³/m³/d for HRT = 9 h with a 50 L/kg COD hydrogen yield (HY) and 40 vol% H₂. Methane production rate (MPR) reaches MPR = 2.6 m³/m³/d with 70 vol% CH₄ at HRT = 2.8 d. The highest H₂ yields are HY = 180 L/kg COD with 53 vol% H₂ (thermophilic, HRT = 48 h). Hyperthermophilic temperatures led to lower HPRs (0.7 m³/m³/d) and MPRs (1.6 m³/m³/d). 53% of Thermoanaerobacterium thermosaccharolyticum as an H₂ producer are found. Discoloration of granular sludge from black to white and granule stability was observed in DF.