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.     

Extreme-thermophilic biohydrogen production from lignocellulosic bioethanol distillery wastewater with community analysis of hydrogen-producing microflora

Extreme-thermophilic biohydrogen production from distillery wastewater was investigated in batch and continuous-mode operation. Hydrogen-producing mixed culture was enriched by repeated batch cultivations. Effect of temperature and pH on biohydrogen yield was investigated in batch experiments. The highest hydrogen yield of 196. mL/g-volatile solids_addded (VS_added) was obtained at 70 °C and pH 7.0 in batch culture. Continuous biohydrogen production was performed in CSTR reactor with yield of 172.0 mL/g-VS_added at HRT (hydraulic retention time) of 4 days. The main metabolic products were acetate, lactate, and ethanol. Community structure of hydrogen-producing microflora was investigated by 16S rRNA gene sequence analysis. The microorganisms involved in both batch and continuous-mode operation were similar and hydrogen production was carried out by a group of extreme-thermophilic bacterial species related to Thermotoga, Coprothermobacter, Caldanaerobacter, Thermobrachium, and Caldicellulosiruptor.     

Microbial characterization of hydrogen-producing bacteria in fermented food waste at different pH values

An anaerobic fermentation of food waste was conducted in a 0.5 L bioreactor incubated at a thermophilic temperature of 55 °C to evaluate the effects of different controlled pH values (5.0, 5.5 and 6.0) on biohydrogen production. Effective biohydrogen production was found at controlled pH 5.5 and 6.0 corresponding to lower lactic acid production compared to pH 5.0. It was demonstrated that biohydrogen production from food waste was pH-dependent with hydrogen yields of 79, 76 and 23 mmol H₂/L-media/d for pH 5.5, 6.0 and 5.0, respectively. Specific microbial determination for Clostridium sp. and total bacteria quantification were carried out by the fluorescent in-situ hybridization (FISH) technique. The number of Clostridium sp. for acclimatized sludge, fermentation broth at pH 5.0, 5.5 and 6.0 were 2.9 × 10⁸, 3.6 × 10⁸, 7.8 × 10⁸ and 5.4 × 10⁸ cells/ml, respectively. The quantification analysis showed that 92% of the total bacteria belonged to Clostridium sp. from clusters I and XI from the sample at controlled pH 5.5. The denaturing gradient gel electrophoresis (DGGE) bands of the sample after heat-treatment, acclimatization and during fermentation indicated the presence of Bacteroidetes, Caloromator australicus sp. and Clostridium sp.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

Selective adaptation of an anaerobic microbial community: Biohydrogen production by co-digestion of cheese whey and vegetables fruit waste

The co-digestion process of crude cheese whey (CCW) with fruit vegetable waste (FVW) for biohydrogen production was investigated in this study. Five different C/N ratios (7, 17, 21, 31, and 46) were tested in 2 L batch systems at a pH of 5.5 and 37 °C. The highest specific biohydrogen production rate of 10.68 mmol H₂/Lh and biohydrogen yield of 449.84 mL H₂/g COD were determined at a C/N ratio of 21. A pyrosequencing analysis showed that the main microbial population at the initial stage of the co-digestion consisted of Bifidobacterium, with 85.4% of predominance. Hydrogen producing bacteria such as Klebsiella (9.1%), Lactobacillus (0.97%), Citrobacter (0.21%), Enterobacter (0.27%), and Clostridium (0.18%) were less abundant at this culture period. The microbial population structure was correlated with the lactate, acetate, and butyrate profiles obtained. Results demonstrated that the co-digestion of CCW with FVW improves biohydrogen production due to a better nutrient balance and improvement of the system's buffering capacity.     

Thermophilic biohydrogen production from sugarcane molasses under low pH: Metabolic and microbial aspects

By-products from sugarcane mills have a considerable energy potential, and therefore have been studied aiming to generate biogas emphasising biohydrogen (bioH₂). Sugarcane molasses, a byproduct from sugar production, are rich in carbohydrates, thus easily biodegraded by anaerobic microorganisms. This study evaluated the production of bioH₂ in unfavorable pH (3.80) using molasses as a feedstock in an anaerobic structured bed reactor (AnSTBR-A) under thermophilic conditions (55 °C). The AnSTBR-A operated with an organic loading rate (OLR) of 60 g L⁻¹ d⁻¹ was able to produce bioH₂ under long-term operation (392 days). The hydrogen yield (HY) was 1.18 mol H₂ mol total carbohydrates⁻¹. The results highlighted HY variation concomitant with metabolite concentrations. The main role to bioH₂ production in AnSTBR-A was acetate + lactate → butyric + bioH₂, with a predominance of the organism belonging to the Thermoanaerobacterium genus.     

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.     

Biohydrogen generation from anaerobic digestion of food waste

Biohydrogen generated from the anaerobic digestion of a synthetic food waste with constant composition and a real food waste collected in Hong Kong were studied. This study aims at using a monoculture to increase biohydrogen production and determining optimum conditions for maximum biohydrogen production. Among the nine bacteria screened for biohydrogen production, Escherichia cloacae and Enterobacter aerogenes produced the largest amount of biohydrogen from the anaerobic digestion of synthetic food waste. The optimum anaerobic digestion conditions were determined: initial pH of 7, a water to solids ratio of 5 (w/w), a mesophilic temperature (37 ± 1 °C), and in the presence of 40 mg/L FeSO₄·7H₂O. Anaerobic digestion at the optimum operating conditions using collected food waste with E. cloacae as the bacterial source was also performed. By adjusting the pH in the range of 5–6, a specific biohydrogen production of 155.2 mL/g of volatile solids (VS) in food waste was obtained.     

Sugarcane vinasse as substrate for fermentative hydrogen production: The effects of temperature and substrate concentration

The present study aimed to evaluate the hydrogen production of a microbial consortium using different concentrations of sugarcane vinasse (2–12 g COD L⁻¹) at 37 °C and 55 °C. In mesophilic tests, the increase in vinasse concentration did not significantly impact the hydrogen yield (HY) (from 1.72 to 2.23 mmol H₂ g⁻¹ COD_influent) but had a positive effect on the hydrogen production potential (P) and hydrogen production rate (R_m). On the other hand, the increase in the substrate concentration caused a drop in HY from 2.31 to 0.44 mmol H₂ g⁻¹ COD_influent in the tests performed at 55 °C with vinasse concentrations from 2 to 12 g COD L⁻¹. The mesophilic community was composed of different species within the Clostridium genus, and the thermophilic community was dominated by organisms affiliated with the Thermoanaerobacter genus. Not all isolates affiliated with the Clostridium genus contributed to a high HY, as the homoacetogenic pathway can occur.     

Enzymatic routes to hydrogen and organic acids production from banana waste fermentation by autochthonous bacteria: Optimization of pH and temperature

The Central Composite Rotational Design (CCRD) was employed to find the optimum pH (5.09–7.91) and temperature (27.1–46,9 °C) for hydrogen production in banana waste (BW) fermentation by autochthonous microbial biomass. The P and Rm ranged between 6.06 and 62.43 mL H₂ and 1.13–12.56 mL H₂.h^?1, respectively. The temperature 37 °C and pH 7.0 were the optimum conditions for P (70.19 mL H₂) and Rm (12.43 mL H₂.h^?1) as predicted by the mathematical model. Fructose and glucose are the primary alternative carbon sources in banana waste-fed batch reactors. The high concentration of lactic acid and H₂ production was associated to Lactobacillus (52–81%) and Clostridium (14–35%). However, the most important finding was about butyric acid (HBu). This acid is the better indicator of hydrogen production than acetic acid (HAc). The pH effected carbohydrates fermentation and organic acids production. The genes encoding the enzymes related to galactose, sucrose, fructose, arabinose and xylose metabolism were predominant.     

Hydrogen production from sulfate- and ferrous-enriched wastewater

The quantitative relationship between sulfate reducing bacteria (SRB) and hydrogen (H₂) production from sulfate (SO₄²⁻) and ferrous [Fe(II)] enriched wastewater was investigated. Both Fe(II) (0–11,600 mg/L) and SO₄²⁻ (0–20,000 mg/L) improved the H₂ production efficiency from wastewater. The H₂ yields were increased up to 1.9 mol H₂/mol glucose in 580–1750 mg Fe(II)/L and 1000–3000 mg SO₄²⁻/L enriched wastewater at pH 5.8–6.2. Quantitative Fluorescence In Situ Hybridization (FISH) analyses revealed that the specific sulfate reducing activities (SSRA) were increased from 0.08 and 0.06 to 0.16 and 0.21 g TS/g SRB h in response to variations in sulfate concentration from 300–20,000 mg/L at pH 5.8 and 6.2, respectively. H₂ production was not influenced by low SSRA (≤0.1 g TS/g SRB h), which was independent of pH variation. The results demonstrated that the SSRA and Fe(II) concentration can significantly influence on the biological H₂ production from SO₄²⁻ and Fe(II) containing wastewater.     

The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations

This study evaluates the effect of pH (4-7) on fermentative biohydrogen production by utilizing three isolated Clostridium species. Fermentative batch experiments show that the maximum hydrogen yield for Clostridium butyricum CGS2 (1.77 mmol/mmol glucose) is achieved at pH 6, whereas a high hydrogen production with Clostridium beijerinckii L9 (1.72 mmol/mmol glucose) and Clostridium tyrobutyricum FYa102 (1.83 mmol/mmol glucose) could be achieved under uncontrolled pH conditions (initial pH of 6.4-6.6 and final pH of 4-4.2). Low hydrogen yields (0-0.6 mmol/mmol glucose) observed at pH 4 are due likely to inhibitory effects on the microbial growth, although a low pH can be thermodynamically favorable for hydrogen production. The low hydrogen yields (0.12-0.64 mmol/mmol glucose) observed at pH 7 are attributed not only to thermodynamically unfavorable, but also metabolically unfavorable for hydrogen production. The relatively high levels of lactate, propionate, or formate observed at pH 7 reflect presumably the high enzymatic activities responsible for their production, together with the low hydrogenase activity, resulting in a low hydrogen production. A correlation analysis of the data from present and previous studies on biohydrogen production with pure Clostridium cultures and mixed microflora indicates a close relation between the hydrogen yield (YH₂) and the (YH₂)/(2(YHAc+YHBu)) ratio, with the observed correlation coefficient (0.787) higher than that (0.175) between YH₂ and the molar ratio of butyrate to acetate (B/A). Based on the (YH₂)/(2(YHAc+YHBu)) ratios observed at different pHs, a control of pH at 5.5-6.8 would seem to be an effective means to enhance the fermentative biohydrogen production.     

Effects of pretreatment of natural bacterial source and raw material on fermentative biohydrogen production

Effects of pretreatment of natural bacterial source and raw material on biohydrogen production in fermentative biohydrogen production process were investigated systematically. Biohydrogen production from stale corn slurry was found to be feasible and effective by A1 (water soak) pretreated compost and B1 (gelatinization) pretreated stale corn. The results were further confirmed by the verification test with working volume of 8 L, in which the maximum hydrogen yield of 262 mL H₂/g-substrate, hydrogen production rate of 39 mL/g h⁻¹ and the corresponding hydrogen content of 50% was observed at fixed substrate concentration of 10 g/L, working pH 5.0-5.5 and 36 ± 1 °C. The effluent was mostly composed of acetate and butyrate. Subsequently, two new hydrogen producing strains were isolated from the effluent sludge in the running bioreactor, and they were preliminarily identified as Clostridium and Enterobacter, respectively, according to the routine screening examinations.     

Dark fermentative biohydrogen production using pretreated Scenedesmus obliquus biomass under an integrated paradigm of biorefinery

In recent times, biohydrogen production from microalgal feedstock has garnered considerable research interests to sustainably replace the fossil fuels. The present work adapted an integrated approach of utilizing deoiled Scenedesmus obliquus biomass as feedstock for biohydrogen production and valorization of dark fermentation (DF) effluent via biomethanation. The microalgae was cultivated under different CO₂ concentration. CO₂-air sparging of 5% v/v supported maximum microalgal growth and carbohydrate production with CO₂ fixation ability of 727.7 mg L^?1 d^?1. Thereafter, lipid present in microalgae was extracted for biodiesel production and the deoiled microalgal biomass (DMB) was subjected to different pretreatment techniques to maximize the carbohydrate recovery and biohydrogen yield. Steam heating (121 °C) in coherence with H₂SO₄ (0.5 N) documented highest carbohydrate recovery of 87.5%. DF of acid-thermal pretreated DMB resulted in maximum H₂ yield of 97.6 mL g^?1 VS which was almost 10 times higher as compared to untreated DMB (9.8 mL g^?1 VS). Subsequent utilization of DF effluent in biomethanation process resulted in cumulative methane production of 1060 mL L^?1. The total substrate energy recovered from integrated biofuel production system was 30%. The present study envisages a microalgal biorefinery to produce biohydrogen via DF coupled with concomitant CO₂ sequestration.     

Anaerobic H2 production at elevated temperature (60 °C) by enriched mixed consortia from mesophilic sources

Two different enriched mixed consortia from mesophilic sources were used for H₂ production from glucose at 60 °C. The variables were initial pH, nitrogen source, iron and sulfate. pH had a crucial effect and iron was slightly positive for the biohydrogen production performance of the mixed culture. On the other hand, H₂ production decreased with the increasing of ammonia, peptone and sulfate concentrations. Metabolic pathways of mixed culture were affected in different ways depending on the differences in microbial community. The PCR-DGGE and sequencing based microbial community analysis revealed that two enriched mixed cultures had different microbial diversity and both culture were dominated mainly by Thermoanaerobacterium species.     

Kinetic analysis of biohydrogen production from anaerobically treated POME in bioreactor under optimized condition

In this study, the biohydrogen production from POME was performed under mesophillic conditions by mixed culture in a 2 L bioreactor using the optimized conditions obtained previously. The effect of controlling pH initially or throughout the fermentation was also examined. The fermentation performance was monitored by comparing P, R_m, λ, and P_s in both systems. In this present study, the reactor system showed higher hydrogen production potential values with the utilization of pH control. Hydrogen production potential was increased two folds when the reactor system was equipped with pH control rather than just fixed the initial pH at 5.8. The biohydrogen production under controlled pH occurred after 7 h fermentation resulting in maximum Ps and Rm of 1.32 L/L POME and 0.144 L/L.h, respectively.     

Specific inhibition of biohydrogen-producing Clostridium sp. after dilute-acid pretreatment of sunflower stalks

Dilute-acid pretreatments are commonly used to solubilize holocelluloses of lignocellulosic materials and represent a promising route to enhance biohydrogen production by dark fermentation. Besides the soluble sugars released, furan derivatives, such as furfural and 5-HMF, as well as phenolic compounds can accumulate in dilute-acid hydrolyzates and that may affect fermentative microbial populations. In this study, biohydrogen production from glucose (5 g VS L⁻¹) in batch tests was investigated in presence of increasing volumes (0% – control, 3.75%, 7.5%, 15% and 35% (v/v)) of dilute acid hydrolyzate generated from sunflower stalks (170 °C, 1 h, 4 g HCl/100 gTS). A sharp decrease of the hydrogen yield was observed from 2.04 mol H₂ mol⁻¹_{eq. hexose initial} in the control to 0 mol H₂ mol⁻¹_{eq. hexose initial} for volumes higher than 15% of added hydrolyzate. Although acetate and butyrate were the main end-products found in the control, ethanol and lactate accumulated accordingly with the increasing addition of hydrolyzate. A clear shift of dominant microbial populations from Clostridium sp. to Sporolactobacillus sp. was concomitantly observed, suggesting a specific inhibition of the biohydrogen-producing bacteria by adding increasing volumes of hydrolyzates.     

Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6

A local bacterial isolate from palm oil mill effluent (POME) sludge, identified as Clostridium butyricum EB6, was used for biohydrogen production. Optimization of biohydrogen production was performed via statistical analysis, namely response surface methodology (RSM), with respect to pH, glucose and iron concentration. The results show that pH, glucose concentration and iron concentration significantly influenced the biohydrogen gas production individually, interactively and quadratically (P < 0.05). The center composite design (CCD) results indicated that pH 5.6, 15.7 g/L glucose and 0.39 g/L FeSO₄ were the optimal conditions for biohydrogen production, yielding 2.2 mol H₂/mol glucose. In confirmation of the experimental model, t-test results showed that curve fitted to the experimental data had a high confidence level, at 95% with t = 2.225. Based on the results of this study, optimization of the culture conditions for C. butyricum EB6 significantly increased the production of biohydrogen.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Development of short chain fatty acid-based artificial neuron network tools applied to biohydrogen production

The biological production of biohydrogen through dark fermentation is a very complex system where the use of an artificial neuron network (ANN) for prediction, controlling and monitoring has a great potential. In this study three ANN models based on volatile fatty acids (VFA) production and speciation were evaluated for their capacity to predict (i) accumulated H₂ production, (ii) hydrogen production rate and (iii) H₂ yield. Lab-scale biohydrogen and VFA production kinetics from a previous study were used for training and validation of the models. The input parameters studied were: time and acetate and butyrate concentrations (model 1), time and lactate, acetate, propionate and butyrate concentrations (model 2), time and the sum of all VFA (model 3) and time and butyrate/acetate (model 4). All models could predict biohydrogen accumulated production, hydrogen production rate and H₂ yield with high accuracy (R² > 0.987). VFA_T is the input parameter indicated for processes using pure cultures, while for complex/mixed cultures a model based on acetate and butyrate is recommended.