Reactor start-up strategy as key for high and stable hydrogen production from cheese whey thermophilic dark fermentation

Using the right start-up strategy can be vital for successful hydrogen production from thermophilic dark fermentation (55 °C), but it needs to be affordable. Hence, three start-up strategies modifying only influent concentration and temperature were assessed in a reactor fed with cheese whey: (i) high temperature (55 °C) and a high organic loading rate (OLR_A - 15 kgCOD m^?3 d^?1) right at the beginning of the operation; (ii) slowly increasing temperature up to 55 °C using a high OLR_A and (iii) slowly increasing temperature and OLR_A up to the desired condition. Strategy (iii) increased hydrogen productivity in 39% compared to the others. The combination of high temperature and low pH thermodynamically favored H₂ producing routes. Synergy between Thermoanaerobacterium and Clostridium might have boosted hydrogen production. Three reactors of 41 m³ each would be needed to treat 3.4 × 10³ m³ year^?1 of whey (small-size dairy industry) and the energy produced could reach 14 MWh month^?1.     

Biological hydrogen production in an anaerobic sequencing batch reactor: pH and cyclic duration effects

An anaerobic sequencing batch reactor (ASBR) was used to evaluate biological hydrogen production from carbohydrate-rich organic wastes. The goal of the proposed project was to investigate the effects of pH (4.9, 5.5, 6.1, and 6.7), and cyclic duration (4, 6, and 8 h) on hydrogen production. With the ASBR operated at 16-h HRT, 25 g COD/L, and 4-h cyclic duration, the results showed that the maximum hydrogen yield of 2.53 mol H₂/mol sucrose_consumed appeared at pH 4.9. The carbohydrate removal efficiency declined to 56% at pH 4.9, which indirectly resulted in the reduction of total volatile fatty acid production. Acetate fermentation was the dominant metabolic pathway at pH 4.9. The concentration of mixed liquor volatile suspended solid (MLVSS) also showed a decrease from nearly 15,000 mg/L between pHs 6.1 and 6.7 to 6000 mg/L at pH 4.9. Investigation of the effect of cyclic duration found that hydrogen yield reached the maximum of 1.86 mol H₂/mol sucrose_consumed at 4-h cyclic duration while ASBR was operating at 16-h HRT, 15 g COD/L, and pH 4.9. The experimental results showed that MLVSS concentration increased from 6200 mg/L at 4-h cyclic duration to 8500 mg/L at 8-h cyclic duration. However, there was no significant change in effluent volatile suspended solid concentration. The results of butyrate to acetate ratio showed that using this ratio to correlate the performance of hydrogen production is not appropriate due to the growth of homoacetogens. In ASBR, the operation is subject to four different phases of each cycle, and only the complete mix condition can be achieved at react phase. The pH and cyclic duration under the unique operations profoundly impact fermentative hydrogen production.     

Effect of inoculum pretreatment on the microbial community structure and its performance during dark fermentation using anaerobic fluidized-bed reactors

The effect of two different inoculum pretreatments, thermal and cell wash-out (A1 and A2, respectively) on the performance of anaerobic fluidized bed reactors for hydrogen production was determined. The reactors were operated for 112 days under the same operational conditions using glucose as substrate at increasing organic loading rates and decreasing hydraulic retention times. Both treatments were effective avoiding methanogenesis. Reactor A2 showed better performance and stability than reactor A1 in each one of the different operational conditions. Cell wash-out treatment produced higher hydrogen volumetric production rates and yields than thermal treatment (7 L H₂/L-d, 3.5 mol H₂/mol hexose, respectively). DGGE analysis revealed that the microbial communities developed were affected by the inoculum treatment. Organisms from the genera Clostridium and Lactobacillus predominated in both reactors, with their relative abundances linked to hydrogen production. Resilience was observed in both reactors after a period of starvation.     

Effects of pH value and substrate concentration on hydrogen production from the anaerobic fermentation of glucose

A series of batch experiments were conducted to investigate the effects of pH and glucose concentrations on biological hydrogen production by using the natural sludge obtained from the bed of a local river as inoculant. Batch experiments numbered series I and II were designed at an initial and constant pH of 5.0–7.0 with 1.0 increment and four different glucose concentrations (5.0, 7.5, 10 and 20 g glucose/L). The results showed that the optimal condition for anaerobic fermentative hydrogen production is 7.5 g glucose/L and constant pH 6.0 with a maximum H₂ production rate of 0.22 mol H₂ mol⁻¹ glucose h⁻¹, a cumulative H₂ yield of 1.83 mol H₂ mol⁻¹ glucose and a H₂ percentage of 63 in biogas.     

Effect of pH on continuous biohydrogen production from liquid swine manure with glucose supplement using an anaerobic sequencing batch reactor

pH is considered as one of the most important factors governing the hydrogen fermentation process. In this project, five pH levels, ranging from 4.4 to 5.6 at 0.3 increments, were tested to evaluate the pH effect on hydrogen production from swine manure supplemented with glucose in an anaerobic sequencing batch reactor system with 16 h of hydraulic retention time (HRT). The optimal hydrogen yield (1.50 mol H₂/mol glucose) was achieved at pH 5.0 when the maximum production rate of 2.25 L/d/L was obtained. Continuous hydrogen production was achieved for over 3 weeks for pH 5.0, 4.7, and 4.4, with no significant methane produced. However, as pH increased to 5.3 and 5.6, methane production was observed in the biogas with concurrent reductions in hydrogen production, indicating that methanogens could become increasingly activated for pH 5.3 or higher. Acetate, propionate, butyrate, valerate, and ethanol were the main aqueous products whose distribution was significantly affected by pH as well.     

Populational and metabolic shifts induced by acetate,butyrate and lactate in dark fermentation

Dark fermentation is subject to inhibition by end products. In this study, the effects of acetate, butyrate and lactate on fermentation routes of glucose were investigated for concentrations ranging from 25 to 400 mM. Whatever the acid considered, an inhibition threshold of hydrogen production was observed at acid concentration as low as 50 mM. 300 mM of acetate, 200 mM of butyrate and 400 mM of lactate were critical concentrations resulting exclusively in lactate production. At these high concentrations, bacterial communities shifted from Clostridiaceae to Lactobacillaceae family after acetate or lactate addition, and to Bacillaceae after butyrate addition. At lower acid concentrations, the nature and the concentration of the added acid shaped metabolic and populational changes. Specifically, Clostridium butyricum was able to grow up to 250 mM, 150 mM and 300 mM of acetate, butyrate and lactate respectively, but was suspected to shift its metabolism towards lactate production.     

Initial pH influences microbial communities composition in dark fermentation of scotta permeate

This study addresses for the first time the influence of initial pH on the evolution of microbial consortia in dark fermentation of scotta permeate, using a high-throughput sequencing approach. Three fermentation phases could be detected: 1) a lag phase with no substantial differences in microbial composition at different initial pH values; 2) an exponential H₂ production phase, accompanied by a general increase of Clostridium genus components and higher incidence of Trichococcus genus at neutral and alkaline pH; 3) a final stationary phase, characterized by a general increase of Bifidobacterium and Lactobacillus genera in all reactors. The initial pH value influenced the relative abundance of Trichococcus at 16–48 h of incubation. The metabolic activity of this genus increased the amount of metabolic precursors of H₂ so that, when pH lowered to 5.4, clostridia in the reactors with initial alkaline pH become more active H₂-producers than those in the others.     

16S rRNA gene based analysis of the microbial diversity and hydrogen production in three mixed anaerobic cultures

To explore of role of microbial diversity and its functionality in commercial bioreactors, three anaerobic microbial communities from Ontario, Canada were characterized using 16S rRNA gene-based, clone library sequencing and terminal restriction fragment length polymorphism (T-RFLP) and compared with the hydrogen (H₂) and methane yields. The T-RFLP method showed more operational taxonomic units than the clone library sequence analysis; however, the two methods showed similar dominant species and relative diversity while Spearman's Rank correlation coefficient (r) values ranged from 0.82 to 0.91. The Chao 1 and Shannon-Wiener indices revealed that the cultures samples have highly diverse microbial communities. Comparatively, cultures from a municipal wastewater treatment plant (CA) showed more diversity than those from facilities treating effluents from a baby food processor and a brewery. Even though culture CA has the highest microbial diversity, low H₂ and methane production yield was attributed to the presence of sulphate reducers, propionate producers and a low percentage of methanogens. This study confirms that the selection of the source of mixed anaerobic cultures plays an important role in H₂ and methane production.     

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.     

Microbial community analysis of mixed anaerobic microflora in suspended sludge of ASBR producing hydrogen from palm oil mill effluent

This study investigated the microbial community of an anaerobic sequencing batch reactor (ASBR) operating at mesophilic temperature under varying hydraulic retention times (HRTs) for evaluating optimal hydrogen production conditions, using palm oil mill effluent (POME) as substrate. POME sludge enriched by heat treatment with hydrogen-producing bacteria was used as inoculum and acclimated with the POME. The microbial community was determined by first isolating cultivable bacteria at each operating HRT and then using polymerase chain reaction (PCR). The PCR products were sequenced and sequence identification was performed using the BLAST algorithm and Genbank database. The findings revealed that about 50% of the isolates present were members of the genus Streptococcus, about 30% were Lactobacillus species and around 20% were identified as species of genus Clostridium. Scanning electron microscopy (SEM) analysis also confirmed the presence of spherical and rod-shaped microbial morphologies in the sludge samples of bioreactor during prolonged cultivation.     

Influence of initial anolyte pH and temperature on hydrogen production through simultaneous saccharification and fermentation of lignocellulose in microbial electrolysis cell

An investigation on the performance of hydrogen production by simultaneous saccharification and fermentation (SSF) in a dual-chamber microbial electrolysis cell (MEC) was carried out to consider different anolyte pH levels and culture temperatures, and the influences of anolyte pH value and culture temperature on changes of current, organic acid and pH value were also evaluated. The maximal hydrogen production rate (HPR) of 2.46 mmol/L/D (hydrogen energy recovery 219.02%) was obtained at the initial anolyte pH of 6.5. Within the range of the tested operation temperatures (30–50 °C), the optimal temperature for hydrogen production by SSF in the MEC systems was 35 °C. Moreover, the contents of organic acids and reducing sugar significantly changed with varying in initial anolyte pH and temperature levels. The result indicates that a low initial anolyte pH value and high culture temperature was beneficial to hydrolysis of cellulose, and a high initial anolyte pH value and a moderate culture temperature to hydrogen production.     

Flux balance analysis of mixed anaerobic microbial communities: Effects of linoleic acid (LA) and pH on biohydrogen production

The internal fluxes of mixed anaerobic cultures fed 2000 mg l⁻¹ linoleic acid (LA) plus glucose at 6 initial pH conditions and maintained at 37 °C were estimated using a flux balanced analysis (FBA). In cultures fed LA at pH 7, less than 8% of the flux was diverted to CH₄. At an initial pH ≥ 5.5, the quantity of glucose removed was greater than 95%; however, at pH 4.5 and 5.0 the quantity consumed were 38% and 75%, respectively. The FBA output showed that the acetogenic H₂-consumers were responsible for more than 20% of the H₂ consumed. Adding LA and decreasing the pH was ineffective in reducing the activity of acetogenic H₂-consumers. As the initial pH decreased, the acetogenic H₂-consuming flux decreased in the presence of 2000 mg l⁻¹ LA. A maximum H₂ yield of 1.55 mol mol⁻¹ glucose consumed (peak hydrogenase flux (R12)) was attained when the acetogenic H₂-consuming flux reached 0.42 mol at a pH of 5.5.     

Advances towards the understanding of microbial communities in dark fermentation of enzymatic hydrolysates: Diversity,structure and hydrogen production performance

Microbial communities involved in hydrogen (H₂) production from enzymatic hydrolysates of agave bagasse were analyzed through 16S rRNA sequencing. Two types of reactor configurations and four different enzymatic hydrolysates were evaluated. Trickling bed reactors led to highly-diverse microbial communities, but low volumetric H₂ production rates (VHPR, maximum: 5.8 L H₂/L-d). On the contrary, well-controlled environments of continuous stirred-tank reactors favored the establishment of low diverse microbial communities composed by Clostridium-Sporolactobacillus leading to high-performance H₂-production (VHPR maximum: 13 L H₂/L-d). Cellulase-Viscozyme and Celluclast-Viscozyme hydrolysates led to the co-dominance of Clostridium and Sporolactobacillus, possibly due to the presence of xylose and hemicellulose-derived carbohydrates. Cellulase hydrolysates were linked to communities dominated by Clostridium, while maintaining low abundance of Sporolactobacillus. Stonezyme hydrolysates favored microbial communities co-dominated by Clostridium-Lachnoclostridium-Leuconostoc. Moreover, contrary to the prevailing theory, it was demonstrated that H₂ production performance was inversely related to microbial diversity.     

Optimizing the production of hydrogen and 1,3-propanediol in anaerobic fermentation of biodiesel glycerol

The conversion of glycerol in biodiesel waste streams to valuable products (e.g. hydrogen and 1,3-propanediol (1,3-PD)) was studied through batch-mode anaerobic fermentation with organic soil as inoculum. The production of hydrogen in headspace and 1,3-PD in liquid phase was examined at different hydrogen retention times (HyRTs), which were controlled by gas-collection intervals (GCIs) and initial gas-collection time points (IGCTs). Two purification stages of biodiesel glycerol (P2 and P3) were tested at three concentrations (3, 5 and 7 g/L). Longer HyRT (longer GCI and longer IGCT) led to lower hydrogen yield but higher 1,3-PD yield. The P3 glycerol at the concentration of 7 g/L had the highest 1,3-PD yield (0.65 mol/mol glycerol_consumed) at the GCI/IGCT of 20 h/65 h and the highest hydrogen yield (0.75 mol/mol glycerol_consumed) at the GCI/IGCT of 2.5 h/20 h), respectively. A mixed-order kinetic model was developed to simulate the effects of GCI/IGCT on the production of hydrogen and 1,3-PD. The results showed that the production of hydrogen and 1,3-PD can be optimized by adjusting HyRT in anaerobic fermentation of glycerol.     

Focusing on the process diagnosis of anaerobic fermentation by a novel sensor system combining microbial fuel cell,gas flow meter and pH meter

Process diagnosis is essential to ensure anaerobic fermentation stable and efficient. Here, a novel sensor system combining microbial fuel cell (MFC), gas flow meter and pH meter was developed to evaluate its feasibility for probing the anaerobic process established on a model high-rate bioreactor. Repeated transient responses of electrical signal, proton concentration, and gas flow rate, were observed subject to external disturbances. The transient response lasted from <1 h to 6 h. In addition, MFC obtained compatible signal variations with other sensors, and biofilm MFC (MFC_Biofilm) resulted in better agreements than control MFC (MFC_Control). These results revealed that 1) the composite sensor system was capable to probe anaerobic process, suggesting a novel approach for process analysis and diagnosis of biogas or biohydrogen production; 2) the variations of sensor signals might provide more valuable information for process diagnosis than sensor signals themselves.     

Assessing the impact of palmitic,myristic and lauric acids on hydrogen production from glucose fermentation by mixed anaerobic granular cultures

The effects of lauric (LUA), myristic (MA), palmitic (PA), and a mixture of myristic:palmitic (MA:PA) acids on hydrogen (H₂) production from glucose degradation using anaerobic mixed cultures were assessed at 37 °C with an initial pH set at 5.0 and 7.131 mM of each acid. The maximum H₂ yield (2.53 ± 0.18 mol mol⁻¹ glucose) was observed in cultures treated with PA. A principal component analysis (PCA) of the by-products and the microbial population data sets detected similarities between the controls and PA treated cultures; however, differences were observed between the controls and PA treated cultures in comparison to the MA and LUA treated cultures. The flux balance analysis (FBA) showed that PA decreased the quantity of H₂ consumed via homoacetogenesis compared to the other LCFAs. The control culture was dominated by Thermoanaerovibrio acidaminovorans (60%), Geobacillus sp. and Eubacterium sp. (28%), while Clostridium sp. was less than 1%. Treatment with PA, MA, MA:PA, or LUA increased the H₂ producers (Clostridium sp. and Bacillus sp.) population by approximately 48, 67, 86, and 86%, respectively.     

Effect of pH control on biohythane production and microbial structure in an innovative multistage anaerobic hythane reactor (MAHR)

An innovative multistage anaerobic hythane reactor (MAHR) which combines an internal biofilm (M_H) and an external up-flow sludge blanket (M_M) was proposed to produce biohythane from wastewater. The effect of pH on its biohythane production and microbial diversity was performed. Results showed that the maximum hydrogen production rate (4.900 L/L/d) was achieved at a pH of 6.0, in comparison to a maximum methane production rate of 10.271 L/L/d at a pH of 6.5. In addition, a suitable hythane (H₂/(H₂+CH₄) of 16.06%) production can be achieved in M_H after the initial pH was adjusted from 7.0 to 6.5, and a relatively high methane yield (271.34 mL CH₄/gCOD) was obtained in M_M. Illumina Miseq sequencing results revealed that decreasing pH led to an increase of the acidogenesis families (Eubacteriaceae, Ruminococcaceae) in M_H and an increase of hydrogenotrophic methanogens (Methanobacteriaceae) in M_M. The Methanosaetaceae gradually occupied a major portion after a long period of recovery. This work demonstrated the unique advantages of MAHR for the biohythane production under optimal pH conditions.     

Early warning indicators and microbial community dynamics during unstable stages of continuous hydrogen production from food wastes by thermophilic dark fermentation

Early warning indicators (EWIs) and microbial community dynamics during thermophilic dark fermentation-based hydrogen production (TFHP) from food wastes were investigated during stable and fluctuating operations in continuous systems. Difference and redundancy analyses revealed the linkage between dominant genera, metabolites and process parameters and characterized the EWIs and mechanism of instability of the TFHP. Besides biogas production rate (GPR), hydrogen proportion and volatile fatty acids (VFAs) concentrations, the potential warning function of the microbial community were observed. Thermoanaerobacterium was the dominant genus which decreased during the unstable stages. Bacillus is another EWI in unstable stages when the value of pH is lower. Lactobacillus and Weissella appeared as OLR increased to 9 g VS L⁻¹ d⁻¹ in the OLR-9 unstable stage. This study demonstrates the key EWIs for monitoring and controlling continuous hydrogen production and its stability.     

Hydrogen production from cassava wastewater using an anaerobic sequencing batch reactor: Effects of operational parameters,COD:N ratio,and organic acid composition

In this work, hydrogen production from cassava wastewater using anaerobic sequencing batch reactors (ASBR) was investigated to determine the optimum number of cycles per day, chemical oxygen demand (COD) loading rate, and COD:N ratio. The system operated at a COD loading rate of 30 kg/m³d and 6 cycles per day provided maximum hydrogen production performance in terms of specific hydrogen production rate (SHPR) (388 ml H₂/g VSS d or 3800 ml H₂/l d) and hydrogen yield (186 ml H₂/g COD removed). The effect of nitrogen supplementation was also studied by adding NH₄HCO₃ into the system at the COD:N ratios of 100:2.2, 100:3.3, and 100:4.4 under the COD loading rate of 30 kg/m³d and 6 cycles per day. The maximum SHPR and hydrogen yield of 524 ml H₂/g VSS d (5680 ml H₂/l d) and 438 ml H₂/g COD removed, respectively, were obtained at the stoichiometric COD:N ratio of 100:2.2. An excess nitrogen was found to promote the productions of higher organic acids and ethanol, resulting in lowering hydrogen production efficiency.     

Predominance of Bacilli and Clostridia in microbial community of biohydrogen producing biofilm sustained under diverse acidogenic operating conditions

Microbial community structure of acidogenic biofilm from long-term operated sequencing batch bioreactor producing biohydrogen was analyzed through culture independent technique. Bioreactor was operated under variable operation and substrate conditions for a period of 1435 days. Phylogenetic distribution showed a significant diversity and illustrated the presence of four dominant operational taxonomic units (OTUs) viz., Bacteroidia, Bacilli, Clostridia, Flavobacteria and Aquificae. Dominance of Clostridia and Bacilli classes were observed each with four OTUs. Majority of OTUs were found to produce fermentative H₂. Even at higher load and under diverse operating conditions bioreactor functioned without any process inhibition which indicates the robustness of sustained microbial community. Community structure of bioreactor was comparatively evaluated with other bioreactor producing H₂, operated with same parent culture and conditions but with different substrates, established the dynamics and shift of microbial diversity which corresponded to diverse substrates used for the bioreactor operation.