Simultaneous analysis of carbohydrates and volatile fatty acids by HPLC for monitoring fermentative biohydrogen production

The production of biohydrogen through anaerobic fermentation has received increasingly attention and has great potential as an alternative process for clean fuel production in the future. The monitoring of the stages of anaerobic fermentation provides relevant information about the bioprocess. The objective of this study is to propose a novel methodology for simultaneous analysis of sucrose, glucose, fructose and volatile fatty acids (VFAs), such as, acetic, propionic, isobutyric and butyric during anaerobic fermentation by using high-performance liquid chromatography (HPLC). The following chromatographic conditions were optimized: column Aminex HPX-87H, mobile phase consisting of H₂SO₄ 0.005 mol/L, flow rate of 1.0 mL/min and temperature of 55 °C. Sucrose, glucose and fructose were analyzed by refractive index detector (RI) while acetic, propionic, isobutyric and butyric acids were analyzed by ultraviolet (UV) detection at 210 nm. Some analytical parameters of validation, such as, linearity, selectivity, repeatability, intermediate precision, limit of detection and quantification, accuracy and robustness were evaluated. The proposed methodology was successfully applied in the determination of substrates and metabolites during different stages of biohydrogen production.     

Hydrogen gas production by electrohydrolysis of volatile fatty acid (VFA) containing dark fermentation effluent

Dark fermentation effluents of wheat powder (WP) solution containing different concentrations of volatile fatty acids (VFAs) were subjected to low voltage (1–3 V) DC current to produce hydrogen gas. Graphite and copper electrodes were tested and the copper electrode was found to be more effective due to higher electrical conductivity. The effects of solution pH (2–7), applied voltage (1–3 V) and the total VFA (TVFA) concentration (1–5 g L⁻¹) on hydrogen gas production were investigated. Hydrogen production increased with decreasing pH and became maximum at pH = 2. Increases in applied voltage and the TVFA concentration also increased the cumulative hydrogen formation. The most suitable conditions for the highest cumulative hydrogen production was pH = 2, with 3 V applied voltage and 5 g TVFA L⁻¹. Up to 110 ml hydrogen gas was obtained with 5 g L⁻¹ TVFA at pH = 5.8 and 2 V applied voltage within 37.5 h. The highest energy efficiency (56%) was obtained with the 2 V applied voltage and 10.85 g L⁻¹ TVFA. Hydrogen production by electrolysis of water in control experiments was negligible for pH > 4. Hydrogen production by electrohydrolysis of VFA containing anaerobic treatment effluents was found to be an effective method with high energy efficiency.     

Degradation of volatile fatty acids in highly efficient anaerobic digestion

To improve the efficiency of anaerobic digestion, we examined the effects of C₂–C₆ volatile fatty acids (VFAs) on methane fermentation, as well as the behavior of VFAs in anaerobic digestion. The VFA concentrations and methane production in anaerobic digestion were increased by pretreatment of waste activated sludge (WAS), such as ultrasonic disintegration, thermal and freezing treatments. The major intermediate products of anaerobic digestion for untreated and pretreated WAS, such as acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate and caproate, were used as substrates and the anaerobic degradation of these was carried out under the same conditions. It was found that decomposition rates of the VFAs (C₂–C₆) with a straight chain (normal form) were greater than those of their respective isomers with a branched chain (iso form). It was shown that the decomposition rates of the iso and normal forms of butyrate were greater than those of valerate and caproate. This was caused by the isomerization between butyrate and isobutyrate which occurred during the digestion process. Anaerobic bacteria in digested sludge converted butyrate to isobutyrate and vice versa by migration of the carboxyl group to the adjacent carbon atom. In addition, inhibition of degradation of the VFAs by acetate in a digester was also examined.     

Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by anaerobic mixed culture fermentation was investigated using batch experiments at 37 °C. Seven varieties of typical individual components of OFMSW including rice, potato, lettuce, lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production potential. Experimental results showed that the boiling treated anaerobic sludge was effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is the most optimal substrate for fermentative hydrogen production compared with proteins, lipids and lignocelluloses. This conclusion was also substantiated by experimental results of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas produced from rice, potato and lettuce were 57–70%, 41–55% and 37–67%.     

Solid-state anaerobic fermentation of spent mushroom compost for volatile fatty acids production by pH regulation

To enhance volatile fatty acid (VFA) production from spent mushroom compost (SMC), the effect of pH from 4.0 to 12.0 was investigated in this study. The results indicated that higher VFA concentration was achieved under alkaline condition compared to acid condition and control. The maximal VFA concentration was 3479.59 mg/L at a pH of 10.0, which was 50.63% higher than the control without pH control. Acetate accounted for more than 50% of total VFAs in all pH values. The NH₄⁺-N and PO₄^3?-P release was in range of 19.56–27.12 mg/g VS and 3.47–15.76 mg/g VS, respectively. Furthermore, the Logistic-based model could well explained the VFA production in this study. Therefore, alkaline fermentation can be considered a promising technology for VFA production from SMC and the optimal pH should be selected as 10.0.     

A comparison of treatment techniques to enhance fermentative hydrogen production from piggery anaerobic digested residues

To accelerate the start-up process and enhance the efficiency of a hydrogen production system, piggery anaerobic digested residues (PADRs) were subjected to several different treatment methods to enrich the hydrogen-producing bacteria. Eight treatment methods were performed on the PADRs, including acid, alkali, heat, drying, ultrasound, aeration, sodium 2-bromoethanesulfonate (BES), and chloroform. The best method was found to be drying at 60 °C for 48 h, which maximised the total biogas production and the hydrogen fraction without causing any methane production. The volatile fatty acids (VFAs) found after the drying treatment were acetate and butyrate, which together accounted for 91.9% of all VFAs, indicating that butyric acid fermentation was established. Due to the drying treatment, the metabolites produced from the biodegradable DOM were utilised more rapidly, more completely, and with the least amount of hard-degradation organic matter content obtained, according to EEM fluorescence spectra. This drying treatment offers a promising method to¹ improve bio-hydrogen production.     

Enhancing biohydrogen production of the alkalithermophile Thermobrachium celere

In this study the effect of different buffering agents, pH control and N₂ sparging on biohydrogen production in Thermobrachium celere was investigated in batch cultivations. Among the tested buffers, none was able to prevent the medium acidification resulting in a premature interruption of the hydrogen production. Controlling the pH helped to sustain the growth, the complete substrate consumption and the H₂ production. However, in these conditions the increase of H₂ partial pressure induced a partial metabolic shift towards ethanol production resulting in a decreased H₂ yield. Analysis of formate accumulation during growth suggests that this compound might play a relevant role in the anabolic routes in T. celere. When frequent N₂ sparging was applied for H₂ removal, together with pH control, the H₂ yield was remarkably enhanced from 2.26 to 3.53 mol H₂/mol glucose, and the maximum H₂ production rate and specific H₂ production rate reached 41.5 mmol H₂/l/h and 142.3 mmol H₂/h/g, respectively. This result suggests that under proper conditions T. celere is able to produce hydrogen at high yield and production rate.     

Enhanced hydrogen and volatile fatty acid production from sweet sorghum stalks by two-steps dark fermentation with dilute acid treatment in between

This study investigated the potential of hydrogen and volatile fatty acid coproduction from two steps dark fermentation with dilute acid treatments of the residual slurry after 1st step fermentation. Sweet sorghum stalks (SS) was used as substrate along with Clostridium thermosaccharolyticum as production microbe. Residual lignocelluloses after 1st step fermentation were treated for 1 h by sulfuric acid concentration of 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5% (w/v) with different reaction temperature of 120, 90 and 60 °C were studied. The optimum severity conditions for the highest yield of products found from the treatment acid concentration of 1.5% (w/v) at 120 °C for 10 g/L of substrate concentration. Experimental data showed that two-step fermentation increased 76% hydrogen, 84% acetic acid and 113% of butyric acid production from single step. Maximum yields of hydrogen, acetic acid and butyric acid were 5.77 mmol/g-substrate, 2.17 g/L and 2.07 g/L respectively. This two-step fermentation for hydrogen and VFA production using the whole slurry would be a promising approach to SS biorefinery.     

Influence of pH, temperature and volatile fatty acids on hydrogen production by acidogenic fermentation

The aim of this work was to study the influence of pH and temperature on acidogenic fermentation and bio-hydrogen production. A centered factorial design was generated with respect to pH (4-6 units) and temperature (26-40 °C), and these conditions were used in batch experiments. Biomass cultivation was conducted in a sequential batch reactor (SBR). A mixed-acidogenic culture enriched from activated sludge and fed with a 9 g/l glucose solution was used in the experiments. At low pH values, hydrogen production was favored when the temperatures were low, a result contrary to those described in literature. Working at higher temperatures reduced the length of the lag phase. Additionally, the hydrogen production rate was increased at these temperatures. These opposite trends indicated that an inhibition effect occurred during the experiment. Hydrogen production was studied by using a response surface methodology, being the highest hydrogen production occurred at pH 5.4 and 26 °C. Regarding to the relationship between the hydrogen and acid production, the hydrogen produced per unit of acetate produced increased as the pH increased. On the other hand, hydrogen produced from other acids was constant and similar to theoretical yields. These values of hydrogen produced per unit of acid produced allowed to estimate the experimental hydrogen production. This result indicated that pH was the most important factor in acidogenic fermentation.     

Upscaling of biohydrogen production process in semi-pilot scale biofilm reactor: Evaluation with food waste at variable organic loads

The present account focuses on upscaling of biohydrogen (H₂) production at semi-pilot scale bioreactor using composite food waste. Experiments were conducted at different organic load (6, 12, 18, 30, 40, 50 and 66 g COD/l) conditions. H₂ production increased with an increasing organic load up to 50 g COD/l (9.67 l/h) followed by 40 g COD/l (6.48 l/h), 30 g COD/l (1.97 l/h), 18 g COD/l (0.90 l/h), 12 g COD/l (0.78 l/h) and 6 g COD/l (0.32 l/h). H₂ production was affected by acidification (pH drop to 3.96) at 66 g COD/l operation due to the excess accumulation of soluble metabolites (5696 mg VFA/l). Variation in organic load of food waste influenced the overall hydrogen production efficiency.     

Integrated Taguchi method and response surface methodology to confirm hydrogen production by anaerobic fermentation of cow manure

In the present study, we evaluated the feasibility of integrating the Taguchi method and the response surface methodology (RSM) to predict and optimize fermentative hydrogen production of cow manure (CM) slurry, a mixture of CM and tap water that was equivalent to 6% of the volatile solid (VS) content. Batch vial tests were first conducted in accordance with an experimental design using the Taguchi method L₁₈ orthogonal array that selected the significant influencing factors (temperature and pH) of hydrogen production, and then the RSM with a central composite design was used for the following experiments based on the aforementioned factors. Finally, fermentation experiments in triplicate were carried out in a 2-L semi-continuously stirred tank reactor (semi-CSTR) with a fixed organic loading rate (OLR), solid retention time (SRT) and varying temperatures and pH under a steady-state operation. Through a series of investigations conducted in this study, our experimental data confirmed that the optimal conditions were 60 °C with pH 5.20 ± 0.21, resulting in hydrogen content (HC) of 54.64 ± 11.45%, volumetric hydrogen production (VHP) of 405.54 ± 193.61 ml-H₂/l/d, and specific hydrogen yield (SHY) of 10.25 ± 4.96 ml-H₂/g-VS. This study demonstrates a good performance of the Taguchi method with pretests and the prediction of the response surfaces methodology. The confirmed experimental results show the behavior of anaerobic fermenters’ treating in significant factors, which will comply with management strategies for treatment of relative organic wastes in the future.     

Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

Metabolic shift and electron discharge pattern of anaerobic consortia as a function of pretreatment method applied during fermentative hydrogen production

We have made an attempt to evaluate the variation in the electron discharge (ED) pattern of anaerobic consortia as a function of pretreatment viz., chemical, heat-shock, acid and oxygen-shock in comparison with untreated mixed consortia during fermentative hydrogen (H₂) production. Experiments were performed with dairy wastewater as substrate using anaerobic mixed consortia as biocatalyst (pretreated individually and in combination). Cyclic voltammetry (CV) elucidated significant variation in the ED pattern of mixed consortia along with H₂ production and substrate degradation (SD) as a function of pretreatment method applied. Higher ED was observed with all pretreated consortia which can be attributed to the stable proton (H⁺) shuttling due to the suppression of methanogenic activity. Oxygen-shock method and untreated consortia showed lower H₂ production and higher SD among the variations studied, while, combined pretreated consortia resulted higher H₂ production and lower SD. Lower ED observed with untreated consortia suggests the H⁺ reduction during methanogenesis rather than the inter-conversion of metabolites, which is presumed to be necessary for H₂ production. ED observed with combined pretreated consortia corroborated well with the observed H₂ production. Redox pairs were visualized on the voltammograms with almost all the experimental variations studied except untreated consortia. The potentials (E₀) of redox pairs observed were corresponding to intracellular electron carriers viz., NAD⁺/NADH (E₀ −0.32 V) and FAD⁺/FADH₂ (E₀ −0.24 V).     

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.     

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.     

Hydrogen production and consumption of organic acids by a phototrophic microbial consortium

Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention because its combustion releases only water, and it can be produced by microorganisms using organic acids as substrates. The aim of this study was to enrich a microbial consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other carbon sources were tested, such as acetate (30 mmol l⁻¹), butyrate (17 mmol l⁻¹), citrate (11 mmol l⁻¹), lactate (23 mmol l⁻¹) and malate (14.5 mmol l⁻¹). The reactors were incubated at 30 °C under constant illumination by 3 fluorescent lamps (81 μmol m⁻² s⁻¹). The cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H₂ l⁻¹ culture for acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H₂ mmol⁻¹ substrate for acetate, butyrate, citrate, lactate and malate, respectively. The consumption of substrates was 43% for acetate, 37% for butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium.     

Critical assessment of biofilm and suspended growth reactor configurations for acidogenic biohydrogen production using wastewater as a function of redox microenvironment

Comprehensive experiments were designed to study the relative function of two bioreactor configurations viz., biofilm and suspended growth for dark-fermentative hydrogen (H₂) production along with wastewater treatment at two varying feed pH conditions (6 and 7). Both the reactors were operated in sequencing batch mode using anaerobic inoculum after pretreatment (combined treatment: acid-shock, heat-shock and chemical-shock). Biofilm system showed efficient H₂ production over the corresponding suspended growth operation at feeding pH 6. VFA profiles visualized high acetate fraction supporting feasible microenvironment for higher H₂ production. Voltammogram profiles visualized significant variation in the bio-electrochemical behavior with the function of operating pH and reactor configuration. It can be inferred from this study that biofilm systems are efficient for H₂ production particularly at acidiophilic microenvironment.     

Performance and composition of bacterial communities in anaerobic fluidized bed reactors for hydrogen production: Effects of organic loading rate and alkalinity

This study evaluated the effects of the organic loading rate (OLR) and pH buffer addition on hydrogen production in two anaerobic fluidized bed reactors (AFBRs) operated simultaneously. The AFBRs were fed with glucose, and expanded clay was used as support material. The reactors were operated at a temperature of 30 °C, without the addition of a buffer (AFBR1) and with the addition of a pH buffer (AFBR2, sodium bicarbonate) for OLRs ranging from 19.0 to 140.6 kg COD m⁻³ d⁻¹ (COD: chemical oxygen demand). The maximum hydrogen yields for AFBR1 and AFBR2 were 2.45 and 1.90 mol H₂ mol⁻¹ glucose (OLR of 84.3 kg COD m⁻³ d⁻¹), respectively. The highest hydrogen production rates were 0.95 and 0.76 L h⁻¹ L⁻¹ for AFBR1 and AFBR2 (OLR of 140.6 kg COD m⁻³ d⁻¹), respectively. The operating conditions in AFBR1 favored the presence of such bacteria as Clostridium, while the bacteria in AFBR2 included Clostridium, Enterobacter, Klebsiella, Veillonellaceae, Chryseobacterium, Sporolactobacillus, and Burkholderiaceae.