Effects of pretreatment in a vortex layer apparatus on the properties of confectionery wastewater and its dark fermentation

Pretreatment prior to anaerobic digestion is an effective option for increasing the biodegradability of organic waste. Vortex layer apparatus (VLA) is considered one of the promising types of equipment for pretreatment. In this work, confectionery wastewater (CW) was pretreated in VLA for 1 and 3 min before dark fermentative hydrogen production in anaerobic upflow biofilters. The pretreatment resulted in a slight increase in soluble chemical oxygen demand (COD), soluble sugars and acetic acid, and a decrease in the concentration of propionic, butyric and caproic acids. Due to the abrasion of steel needles in VLA, the concentration of iron in the pretreated CW increased by 2.57 times. Hydraulic retention time in anaerobic upflow biofilters was gradually reduced from 5.6 to 1.8 and 1.3 days, which corresponded to organic loading rate of 2.0, 6.3 and 8.8 kg COD/(m³ day). Although the highest hydrogen yield (96.2 ± 8.1 ml/g COD) was obtained for non-pretreated CW, the pretreatment contributed to a significant increase in methane yield (39.2 ± 2.5 ml/g COD), possibly due to higher iron content (1.8 ± 0.3 mg/L). The highest energy production rate (4407 J/(L day)) was achieved after 3 min CW pretreatment. Thus, pretreatment in VLA can be a promising method for improving the biohythane production process.     

Enhancement of fermentative biohydrogen production from textile desizing wastewater via coagulation-pretreatment

A real textile desizing wastewater (TDW) was coagulation-pretreated to enhance its potential of biohydrogen production. Batch fermentation showed that the hydrogen production was efficiently enhanced (550 and 120% increments for hydrogen production rate and hydrogen yield, respectively) and the production performance was substrate-concentration dependent. A peak hydrogen production rate of 3.9 L/L-d and hydrogen yield of 1.52 mol/mol hexose were obtained while using coagulant GGEFloc-653 at a dosage of 1 g/L to pretreat TDW with the concentration of 15 g total sugar/L. The coagulation-pretreatment could have butyrate-type fermentation with high biohydrogen production and the removed some toxic materials that might drive the metabolic pathways to those not favoring biohydrogen production. Based on the data obtained, strategies to operate the coagulation and biohydogen fermentation are suggested. Moreover, fermentation effluent utilization such as for two-stage biogas production and further biohythane (a mixture of H₂ and CH₄) generation are also elucidated.     

Dark fermentation of starch factory wastewater with acid- and base-treated mixed microorganisms for biohydrogen production

Biohydrogen (Bio-H₂) can be produced from starch factory wastewater and mixed microorganisms using dark fermentation. Acidic and basic chemicals were used to treat the microorganisms to select the hydrogen (H₂)-producing culture. The experiment used a 120 mL bioreactor at 35 °C and the operation commenced with the initial pH level of wastewater in the pH range 4–7 in batch mode. The bacteria:chemical oxygen demand (COD) ratio was 0.2. The initial pH level of the wastewater in the fermentation process affected the H₂ yield and the specific hydrogen production rate (SHPR). For acid-treated bacteria, the maximum H₂ yield and SHPR were produced at an initial pH of 6.5. The maximum H₂ yield and SHPR were 138 mL/g COD degraded and 7.42 mL/g cells?h, respectively. For the base-treated bacteria, the maximum H₂ yield and SHPR were produced at initial pH of 6.5 and pH 7, respectively. The maximum H₂ yield and SHPR were 182 mL/g COD degraded and 25.60 mL/g cells?h, respectively. The COD degradation efficiency levels were 16 and 20% for acid- and base-treated bacteria, respectively. The digested wastewater remained acidic at pH 4.79–4.83. Throughout the study, no methane gas was observed in the gas mixture produced.     

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H₂/g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Kinetic analysis of biohydrogen production from complex dairy wastewater under optimized condition

Present work describes a kinetic analysis of various aspects of biohydrogen production in batch test using optimized conditions obtained previously. Monod model and Logistic equation have been used to find growth kinetic parameters in batch test under uncontrolled pH. The values of μ_m, K_s, and X_m were 0.64 h⁻¹, 15.89 g-COD L⁻¹, and 7.26 g-VSS L⁻¹, respectively. Modified Leudeking-Piret and Michaelis–Menten equation corroborates a flux of energy to hydrogen production pathway and energy sufficiency in the system. Modified Gompertz equation illustrates that the overall rate and hydrogen yield at 15 g-COD L⁻¹ was higher compared to a dark fermentation of other wastewaters. Besides, Andrew's equation also suggests that since the higher value of K_I (19.95 g-COD L⁻¹), k (255 mL h⁻¹ L⁻¹) was not inhibited at high S. The experimental results implied that the entire products during the fermentation process were growth and substrate degradation associated. The result also confirms that the acetate and butyrate were substantially used for hydrogen production in acidogenic metabolism under uncontrolled pH.     

Techno-economic evaluation of biohydrogen production from wastewater and agricultural waste

The world is facing serious climate change caused in part by human consumption of fossil fuel. Therefore, developing a clean and environmentally friendly energy resource is necessary given the depletion of fossil fuels, the preservation of the earth's ecosystem and self-preservation of human life. Biological hydrogen production, using dark fermentation is being developed as a promising alternative and renewable energy source, using biomass feedstock. In this study, beverage wastewater and agricultural waste were examined as substrates for dark fermentation to produce clean biohydrogen energy.     

Feasible pretreatment of textile wastewater for dark fermentative hydrogen production

In this study, the yield of hydrogen production was investigated under different feedstock pretreatment conditions. The feedstock for dark fermentative hydrogen production was textile wastewater which was obtained from the de-sizing process in a textile factory, located in northern Taiwan. The wastewater was pretreated with activated carbon, cation exchange resin or was not pretreated before being fed into the batch bottles. Biohydrogen production was carried out in a batch reactor with the sludge of mixed-culture using the feedstock from the pretreated wastewater. The sludge was obtained from the Taichung municipal wastewater treatment plant. The yield of hydrogen production using the two pretreatment methods or non – treatment were compared.     

Dark fermentative hydrogen production from food waste: Effect of biomass ash supplementation

This work aimed to investigate the effects of supplementing two distinct types of ash, namely fly ash (FA) and bottom ash (BA) on the dark fermentation (DF) process of food waste (FW) for H₂ production. Both types of biomass combustion ash (BCA) were collected in an industrial bubbling fluidized bed combustor, using residual forest biomass as fuel. Results indicated that adding BCA at different doses of 1, 2 and 4 g/L could effectively enhance H₂ generation when compared to the control test without BCA addition. This stimulatory effect was attributed to the crucial role of metal elements released from BCA such as sodium, potassium, calcium, magnesium, and iron in the provision of buffering capacity and inorganic nutrients for the functioning of hydrogen-forming bacteria. The highest H₂ yield of 169 mL per g of volatile solids (VS) were obtained by adding only a small amount of BA (1 g/L) to the reactive system, representing a significant increment of 1070% compared to the control reactor. Furthermore, a significant decrease in the bacterial lag phase time from 26 h to 2.7 h, as well as about a 12-fold increase in the energy recovery as H₂ gas was observed at BA dosage of 1 g/L in comparison with the control reactor. Overall, this study suggested that a proper addition of BCA could promote the DF process of FW and enhance biohydrogen production.     

Lactate wastewater dark fermentation: The effect of temperature and initial pH on biohydrogen production and microbial community

Biohydrogen production using dark fermentation (hydrolysis and acidogenesis) is one of the ways to recover energy from lactate wastewater from the food-processing industry, which has high organic matter. Dark fermentation can be affected by the temperature, pH and the microbial community structure. This study investigated the effects of temperature and initial pH on the biohydrogen production and the microbial community from a lactate wastewater using dark fermentation. Biohydrogen production was successful only at lower temperature levels (35 and 45 °C) and initial pH 6.5, 7.5 and 8.5. The highest hydrogen yield (0.85 mol H₂/mol lactate consumed) was achieved at 45 °C and initial pH 8.5. The COD reduction achieved by fermenting the lactate wastewater at 35 °C ranged between 21 and 30% with the maximum COD reduction at pH 8.5, and at 45 °C, the COD reduction ranged between 12 and 21%, with the maximum at pH 7.5. At 35 °C, the lactate degradation ranged between 54 and 95%, while at 45 °C, it ranged between 77 and 99.8%. 16S rRNA sequencing revealed that at 35 °C, bacteria from the Clostridium genera were the most abundant at the end of the fermentation in the reactors that produced hydrogen, while at 45 °C Sporanaerobacter, Clostridium and Pseudomonas were the most abundant.     

Fermentative biohydrogen production: Evaluation of net energy gain

Most dark fermentation (DF) studies had resorted to above-ambient temperatures to maximize hydrogen yield, without due consideration of the net energy gain. In this study, literature data on fermentative hydrogen production from glucose, sucrose, and organic wastes were compiled to evaluate the benefit of higher fermentation temperatures in terms of net energy gain. This evaluation showed that the improvement in hydrogen yield at higher temperatures is not justified as the net energy gain not only declined with increase of temperature, but also was mostly negative when the fermentation temperature exceeded 25 °C. To maximize the net energy gain of DF, the following two options for recovering additional energy from the end products and to determine the optimal fermentation temperature were evaluated: methane production via anaerobic digestion (AD); and direct electricity production via microbial fuel cells (MFC). Based on net energy gain, it is concluded that DF has to be operated at near-ambient temperatures for the net energy gain to be positive; and DF + MFC can result in higher net energy gain at any temperature than DF or DF + AD.     

Optimization of culture conditions for biohydrogen production from sago wastewater by Enterobacter aerogenes using Response Surface Methodology

Sago wastewater (SWW) causes pollution to the environment due to its high organic content. Annually, about 2.5 million tons of SWW is produced in Malaysia. In this study, the potential of SWW as a substrate for biohydrogen production by Enterobacter aerogenes (E. aerogenes) was evaluated. Response Surface Methodology (RSM) was employed to find the optimum conditions. From preliminary optimization, it was found that the most significant factors were yeast extract, temperature, and inoculum size. According to Face Centered Central Composite Design (FCCCD), the maximum hydrogen concentration and yield were 630.67 μmol/L and 7.42 mmol H₂/mol glucose, respectively, which is obtained from the sample supplemented with 4.8 g/L yeast extract concentration, 5% inoculum, and incubated at the temperature of 31 °C. Cumulative hydrogen production curve fitted by the modified Gompertz equation suggested that H_max, R_max, and λ from this study were 15.10 mL, 2.18 mL/h, and 9.84 h, respectively.     

Feasibility of biohydrogen production at low temperatures in unbuffered reactors

Feasibility of biohydrogen production by dark fermentation at two temperatures (22 °C and 37 °C) in unbuffered batch reactors was evaluated using heat-treated compost as inocula and sucrose as substrate, without any initial pH adjustment or inorganic nutrient supplements. Gas production was quantified by two different pressure release methods – intermittent pressure release (IPR) and continuous pressure release (CPR). Hydrogen production (47.2 mL/g COD/L) and sucrose-to-hydrogen conversion efficiency (53%) were both found to be highest at the lower temperature and IPR conditions. Hydrogen production was higher at the lower temperature irrespective of the pressure release condition. The high yield of 4.3 mol of hydrogen/mole of sucrose obtained in this study under IPR conditions at 22 °C is equivalent to or better than the literature values reported for buffered reactors. Even though literature reports have implied potential inhibition of hydrogen production at high hydrogen partial pressures resulting from IPR conditions, our results did not show any negative effects at hydrogen partial pressures exceeding 5.0 × 10⁴ Pa. While our findings are contrary to literature reports, they make a strong case for cost-effective hydrogen production by dark fermentation.     

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.     

The CRISPR technology: A promising strategy for improving dark fermentative biohydrogen production using Clostridium spp.

Generating hydrogen gas (H₂) using the dark fermentation method has attracted much attention due to its lower energy requirement and environmental friendliness. However, producing a high yield of bio-H₂ is as challenging as ever due to low energy conversion by microorganisms. In this respect, the advancement of genome editing tools including the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology could overcome the established maximum ceiling of product yield. To date, CRISPR-Cas systems, particularly those based on Type II CRISPR-Cas9 and Type V CRISPR-Cas12, are widely used in manipulating novel bacteria to improve the yield of specific biofuel. However, studies using the CRISPR-Cas technology for improving bio-H₂ production remain scarce. Understanding the metabolic pathways of Clostridium spp. is essential for using the CRISPR-Cas technology Thus, this review highlighted the state-of-the-art in CRISPR-Cas systems for bacterial genome editing while paying attention to bioprocess optimization strategies for modulating the biohydrogen production.     

Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H₂/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H₂/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.     

Improvement of biohydrogen production from glycerol in micro-oxidative environment

Glycerol is a highly available by-product generated in the biodiesel industry. It can be converted into higher value products such as hydrogen using biological processes. The aim of this study was to optimize a continuous dark fermenter producing hydrogen from glycerol, by using micro-aerobic conditions to promote facultative anaerobes. For that, hydrogen peroxide (H₂O₂) was continuously added at low but constant flow rate (0.252 mL/min) with three different inlet concentrations (0.2, 0.4, and 0.6% w/w). A mixture of aerobic and anaerobic sludge was used as inoculum. Results showed that micro-oxidative environment significantly enhanced the overall hydrogen production. The maximum H₂ yield (403.6 ± 94.7 mmolH₂/molGly_consumed) was reached at a H₂O₂ concentration of 0.6% (w/w), through the formate, ethanol and butyrate metabolic pathways. The addition of H₂O₂ promoted the development of facultative anaerobic microorganisms such as Klebsiella, Escherichia-Shigella and Enterococcus sp., likely by consuming oxygen traces in the medium and also producing hydrogen. Despite the micro-oxidative environment, strict anaerobes (Clostridium sp.) were still dominant in the microbial community and were probably the main hydrogen producing species. In conclusion, such micro-oxidative environment can improve hydrogen production by selecting specific microbial community structures with efficient metabolic pathways.     

Effect of pH shock on single-stage biohythane production using gel-entrapped anaerobic microorganisms

A novel single-reactor system having entrapped anaerobic microorganisms has been developed to co-produce H₂ and CH₄. pH is one of the key operating and environmental parameters affecting the performance of a bio-system. This work aimed to investigate the pH shock effects on the novel biohythane system. The experiments were suddenly changing the original cultivation pH value of 6 into 4, 5, 7 or 8 for 4 h. The results indicate that a short pH shock could be used to regulate H₂/CH₄ composition without notably affecting biogas yield and chemical oxygen demand (COD) removal. Peak biohythane production was obtained after the pH shock to 8, having H₂/CH₄ yields of 11.5 ± 1.6/44.8 ± 3.1 mL/g COD. During pseudo steady-state conditions of effective cultivation periods, the values of H₂ content in biohythane and COD removal efficiency were in ranges of 20–39% and 71–79%, respectively. The significances and applications of the experimental results have been discussed. The novelty of this work is elucidating a less-discussed field-operation problem of pH perturbances for a newly-developed biohythane system.     

Dark fermentative hydrogen production from simple sugars and various wastewaters by a newly isolated Thermoanaerobacterium thermosaccharolyticum SP-H2

The hydrogen-producing bacterium SP-H2 was isolated from a thermophilic acidogenic reactor inoculated with municipal sewage sludge and processing a carbohydrate-rich simulated food waste. Based on the 16S rRNA gene sequence, the bacterium was identified as Thermoanaerobacterium thermosaccharolyticum. The maximum growth rate was observed at 55–60 °C and pH 7.5. The H₂-producing activity of the bacterium was studied using mono-, di- and tri-saccharides related to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose and arabinose), as well as using real wastewaters (cheese whey, confectionery wastewater, sugar-beet processing wastewater). The highest H₂ yield was observed during dark fermentation (DF) of maltose (1.91 mol H₂/mol hexose or 77.8 mmol H₂/L). The maximum H₂ production rate was observed during DF of xylose (13.3 ml H₂/g COD/h) and cellobiose (2.47 mmol H₂/L/h). The main soluble metabolite products were acetate, ethanol and butyrate. The acetate concentration had a statistically significant positive correlation with the H₂ content in biogas and the specific H₂ yield. Based on the results of the correlation analysis, it was tentatively assumed that in the formic acid (mixed-acid) type fermentation, the rate of H₂ production was higher than in the butyric acid type fermentation. With regard to real wastewater, cheese whey and confectionery wastewater were distinguished by a higher H₂ yield (152 ml H₂/g COD) and H₂ production rate (0.57 mmol H₂/L/h), respectively. The highest concentrations of confectionery wastewater and cheese whey, at which the DF process took place, were 5915 and 7311 mg COD/L, respectively. At the same time, SP-H2 dominated in the microbial community, despite the presence of indigenous microorganisms in wastewater. Thus, T. thermosaccharolyticum SP-H2 is a promising strain for DF of carbohydrate-rich unsterile wastewater under thermophilic conditions.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.