Butyric Acid-type Fermentation Research Articles

The synergistic hydrogen production of bicellular fermentation systems and fluid dynamics simulation in reactor under stirring

The effect of stirring (200–800 rpm) on the synergistic fermentation hydrogen production by bicellular system consisted of Bacillus cereus (A) and Brevundimonas naejangsanensis (B) was investigated. Results indicated that hydrogen yield is the highest (1.36 mol H2/mol glucose) at 800 rpm for bicellular system and its efficiency is 1.3 and 1.4 times higher than the highest value obtained from the single fermentation of Strain A at 800 rpm and Strain B at 400 rpm, respectively. Butyric acid-type fermentation is dominated during the co-cultured fermentation at preferred stirring speed. Results via computational fluid dynamics (CFD) simulations showed that the area-weighted average value of velocity (m/s) on the cross section (y = 4.365 mm) is 0.104, 0.198 and 0.358 for the co-cultured fermentation under different stirring speeds, respectively. Major hydrodynamic parameters increased with the increasing stirring speed. This preliminarily revealed that hydrogen yield is seriously limited by the main accumulated metabolites.

Insights into the Effect of Rhamnolipids on the Anaerobic Fermentation and Microalgae Lipid Production of Waste Activated Sludge: Performance and Mechanisms

In the last few years, the production of waste activated sludge (WAS) has increased dramatically, and the treatment and disposal have attracted widespread attention. In this study, rhamnolipids were used to improve the efficiency of hydrogen production from WAS, and the fermentation solution was used as a substrate by lipid-producing microalgae to recover resources and generate clean energy. Results showed that rhamnolipids had the best effect among several surfactants in promoting hydrogen production, and the hydrogen production of WAS added with 0.4 g/g TS rhamnolipids reached 178.48 ± 6.07 mL/g TS, which improved by 13.53 times compared to the control group. The content of SCOD, soluble protein, and soluble polysaccharide in WAS fermentation solution with the addition of 0.4 g/g TS rhamnolipid increased by 9.19, 11.46, and 42.28 times, respectively, indicating that rhamnolipid could improve the hydrolysis of WAS, which led to an increase in the efficiency of hydrogen production. And three-dimensional excitation–emission matrix fluorescence spectroscopic analysis showed that the intensity of dissolved organic matter increased, suggesting a rise in soluble microbial metabolites. The analysis of the composition of volatile fatty acids in the fermentation solution showed that the type of hydrogen production was mainly butyric acid-type fermentation hydrogen production with high concentrations of n-butyric acid (9330.14 ± 525.33 mg/L) and acetic acid (6324.07 ± 80.67 mg/L). In addition, analysis of the microbial community structure of the WAS fermentation hydrogen production system showed that the dominant genus was Clostridium sensu stricto. Microalgae can utilize acetic acid, ethanol, NH4+-N, PO43–-P, dissolved proteins, and dissolved polysaccharides in the fermentation solution to grow and produce lipids. The biomass and lipid productivity of microalgae cultured by fermentation solution reached 3.16 ± 0.58 g/L and 356.86 ± 4.91 mg/L/day, respectively. The microalgae contained 84.57 ± 4.33% saturated fatty acid content, which was more favorable for biodiesel production. This study not only successfully utilized rhamnolipids to promote hydrogen production from WAS but also coupled microalgae lipid production system, further laying the foundation for realizing the resource utilization of waste resources and clean energy production.

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.

Effect of continuous micro-aeration on hydrogen production by coal bio-fermentation

ABSTRACT In order to study the effect of micro-aeration on hydrogen production from coal, bituminous coal D from the Yima mining area was selected. An experiment to simulate hydrogen production by bio-fermentation was carried out with mine water as the source of bacteria. The results show that with an increasing aeration rate, the hydrogen production continuously decreased. At the end of gas production, the total hydrogen production under aeration rates of 0.0015 L/h, 0.015 L/h and 0.15 L/h was 3.60 mL/g, 1.50 mL/g and 0.58 mL/g, respectively. The molar concentrations of volatile fatty acids (VFAs) in the reaction liquid increased continuously, and the pH decreased overall. The COD content of the liquid phase first increased and then decreased. The COD degradation rates of three different aeration rates at the end of hydrogen production were 43.67%, 16.26% and 5.87%, respectively. With the increasing aeration rate, the dominant hydrogen producing bacteria during bio-fermentation gradually changed from Clostridium to Propionibacterium, and the type of fermentation also changed from butyric acid-type fermentation to propionic acid-type fermentation. In conclusion, the experimental results provide a theoretical basis for exploring hydrogen production via coal bio-fermentation under continuous micro-aeration.

Effects of different temperatures and pH values on volatile fatty acids production during codigestion of food waste and thermal-hydrolysed sewage sludge and subsequent volatile fatty acids for polyhydroxyalkanoates production

The effects of temperature (35 °C and 55 °C) and pH (uncontrolled, 7 and 10) on volatile fatty acid (VFA) yields from anaerobic codigestion of food waste, and thermal-hydrolysed sewage sludge were investigated in this study. The results revealed that optimal conditions for VFA production occurred at 35 °C at pH 7 and at 10 and 55 °C at pH 7. The dominant bacterial genera associated with VFA production significantly differed when the temperature and pH were altered, including Prevotella, Lactobacillus, Bifidobacterium Megasphaera, Clostridium XlVa, and Coprothermobacter. A temperature of 35 °C at pH 7 favoured mixed acid-type fermentation, while a temperature of 35 °C at pH 10 and 55 °C at pH 7 favoured butyric acid-type fermentation. The maximal polyhydroxyalkanoate content accounted for 54.8% of the dry cell at 35 °C with pH 7 fermentative liquids and comprised 58.9% 3-hydroxybutyrate (3HB) and 41.1% 3-hydroxyvalerate (3HV).

Chloroacetaldehyde removal by zero valent iron enhanced hydrolytic acidification pretreatment

Chloroacetaldehyde is a typical refractory toxic petrochemical wastewater to environment, whereas conventional treatment strategies in the wastewater treatment plants are inapplicable to transforming it into nontoxic forms. Therefore, we explored zero valent iron enhanced hydrolytic acidification solution to tackle this problem. Experimental results indicated that zero valent iron enhanced hydrolytic acidification technology possessed the capability to chloroacetaldehyde detoxication, 20.23% and 19.32% increasing dechlorination ratio achieved at initial CAE concentration of 50 and 100 mg/L with 5 g/L ZVI at 48 h. Moreover, ZVI reduced cell death resulted from 0.31, to 1.59 DNA/g VSS reduced at aforementioned parameters. Also, ZVI did not significantly affect predominant butyric acid-type fermentation, but it facilitated the total volatile fatty acids more 43.10, 26.06 and 25.39 mg COD/g VSS produced at 50 and 100 mg/L with at 48 h, particularly, it enhanced the yields of short-chain fatty acid Ac which can be directly used by microbes.

Bio-hydrogen production by a new isolated strain Rhodopseudomonas sp. WR-17 using main metabolites of three typical dark fermentation type

In this work, a new strain WR-17 was isolated for photo-fermentative hydrogen production and its hydrogen production capacity was investigated by utilizing main liquid byproducts of three dark fermentation types in batch culture. Experimental results indicated that strain WR-17 was identified as genus Rhodopseudomonas and maximum hydrogen yield of 2.42 mol H2/mol acetate was obtained when the acetate was used as sole carbon source. Strain WR-17 had an excellent ability of using mixed short chain acids of three typical fermentations such as acetate and ethanol, acetate and butyrate, acetate and propionate. Result demonstrated that the metabolites of butyric acid-type fermentation as substrate is fitting to produce hydrogen and maximum cumulative hydrogen volume of 2156 ml/L-medium was obtained when acetate of 30 mmol/L and butyrate of 15 mmol/L were used. Therefore, butyric acid-type fermentation has great potential for further obtaining high hydrogen yield by the combining photo-fermentation.

Effect of temperature on the fermentation of starch by two high efficient H2 producers

The batch fermentative hydrogen production using starch was conducted to investigate the influence of temperature (25 °C-40 °C) on the co-cultures system of Bacillus cereus ATCC 14579 (T) [Strain A] and Brevundimonas naejangsanensis BIO-TAS2-2(T) [Strain B]. The results showed that the optimal temperature for hydrogen production was 35 °C in both the sole and the mixed culture fermentation. At this temperature, the co-culturing system increased the substrate consumption rate by 34 % and 70 % compared with the Strain A and Strain B fermentation, respectively. The co-culturing system enhanced the hydrogen production yield by 52 % and 62 % compared with the Strain A and Strain B fermentation, respectively. The analysis of soluble metabolites during the fermentation process indicated that the mixed acid fermentation conducted in two strains and co-culturing system was dominated by butyric acid type fermentation at the optimal temperature. The Formate pathway probably exists in Strain A, and the PFOR pathway probably exists in Strain B. Both strains A and B play a synergistic role in hydrogen production at 35 °C using starch as substrate.

Acidogenic properties of carbohydrate-rich wasted potato and microbial community analysis: Effect of pH

Vegetable waste is one of the major organic solid residues available for sustainable biogas production. The aim of this study was to investigate the possibility and optimal controlling strategy for acidogenic fermentation of wasted potato (WP). Three leaching bed reactors (LBRs) were operated at various pH values (6.0, 7.0 and 8.0) with an organic loading rate (OLR) of 6.7g volatile solid/(L·d) and hydraulic retention time of 6d. Butyric acid-type fermentation with butyric acid as predominant volatile fatty acid (VFA) was observed with a concentration and proportion (of total VFAs) of butyric acid, which were 7.8g/L, 49.7 % and 9.6g/L and 52.2 % at pH 6.0 and 7.0, respectively. Conversely, at pH 8.0, mixed acid-type fermentation was observed with acetic and butyric acid as the major VFAs. Control experiment without pH manipulation didn't perform well in VFAs production at first 6 days and then VFAs concentration increased as pH value was adjusted to 8. It was indicated that the inhibition was caused by high undissociated VFAs concentration due to low pH and the VFAs production could be improved through pH control strategy to regulate the undissociated VFAs concentration. According to the bacterial analysis, the microbial community was diverse and Firmicutes were the most important bacteria at different pH conditions. Therefore, the results suggested that a process of pH control might be feasible for stable and efficient acidogenic fermentation.

Anaerobic co-digestion of food waste/excess sludge: substrates - products transformation and role of NADH as an indicator

The process of anaerobic co-digestion is vital importance to resource recovery from organic solid wastes such as food waste and municipal sludge. However, its application is hindered by the limited understanding on the complex substrates-products transformation reactions and mechanisms therein. In this study, food waste (FW) and excess sludge (ES) from municipal wastewater treatment were mixed at various ratios (ES/FW 5:0, 4:1, 2:1, 1:1, 1:2, 1:4, w/w), and the co-digestion process was studied in a batch test. The consumption of substrates including soluble proteins and carbohydrates, the variation in the intermediates such as various volatile fatty acids, and the production of hydrogen and methane gases were monitored. The results suggested that 4:1 was likely the optimal ratio where substrates were consumed and biogas generated efficiently, whereas 1:2 and 1:4 caused severe inhibition. Fermentation of ES alone produced mainly acetic and propionic acid, while the addition of FW led to butyric acid type fermentation. Intermediates in the fermentation liquid were tentatively identified, and the levels of NADH quantified using 3D-excitation/emission fluorescence spectrometry. One class of the intermediates, tryptophan-like proteins were correlated to the butyric acid accumulation in ES/FW mixtures, and NADH level was proposed as an indicator of VFAs production activities.

Effects of certain long-chain fatty acid combinations on the ruminal microbe species relating to fermentation type in vitro

This experiment was conducted to investigate the effects of certain long-chain fatty acid combinations on ruminal microbial community in vitro. Three cows fitted with permanent ruminal cannulas were used to provide rumen liquor for the in vitro trail. The treatments were fatty acid combinations of stearic acid, oleic acid, linoleic acid and linolenic acid, which respectively were group A (calcium palmitate, control), B (1.5%, 1.0%, 0.5% and 1.5%, acetic type fermentation), C (1.5%, 1.0%, 1.5% and 1.0%, propionic acid type fermentation), D (1.0%, 1.5%, 1.5% and 0.5%, butyric acid type fermentation). Three goats fitted with rumen cannula were used to provide the culture medium was collected for the measurement of the rumen microorganism in vitro culture. Culture medium was collected at 0, 3, 6, 12, 18 and 24 h. The results demonstrated that, except for Ruminococcus albus, all the other bacterial genus had significant differences between groups (P less than 0.05). The Fibrobacter succinogenes and Ruminococcusflavefaciens, Clostridium proteoclasticum and Ruminobacter amylophilus were higher in group B; Butyrivibrio fibrisolvens, Megasphaera elsdenii and Ruminococcus bromii were higher in group C; while Pseudobutyrivibrio ruminis was higher in group D. Different higher species in different treatments described above related to their fermentation type. It was also observed that, bacteria were the dominant flora in rumen and Megasphaera elsdeniiwere the dominant species in bacterial fraction no matter what the treatment is. It was therefore concluded that, certain fatty acid combinations have remarkable effects on the amount of ruminal specific bacteria species,mainly related to their fermentation type.

Pretreatment of Corn Stover with Diluted Nitric Acid for the Enhancement of Acidogenic Fermentation

In this study, dilute nitric acid was selected as the catalyst to pretreat corn stover. The effects of nitric acid pretreatments on the efficiency of acidogenic fermentation and the acidogenic characteristic of prehydrolysates were evaluated. The results showed that about 97% hemicelluloses were recovered at the optimal condition (150 °C, 0.6% HNO3, 2 min). Acidogenic fermentation of prehydrolysate B (the highest yield of total sugar) obtained at the optimal condition proved to be more efficient compared with prehydrolysate A (no inhibitor) and prehydrolysate C (the highest yield of inhibitors), the VFAs concentration can reach the highest level, and the distribution of VFAs exhibited butyric acid type fermentation; this was more suitable for subsequent methanogenic fermentation. This research provided an effective and suitable method for accelerating biogas production from corn stover.

Two-phase anaerobic digestion of municipal solid wastes enhanced by hydrothermal pretreatment: Viability, performance and microbial community evaluation

The viability of hydrothermal pretreatment (HTP) to improve the efficiency of biogas production from municipal solid wastes (MSWs) is uncertain because there is always a trade-off between organic matters hydrolysis and recalcitrant melanoidins formation when applying HTP on different solid wastes. In this study, MSWs including waste activated sludge, fruit and vegetable residues, and kitchen wastes, with and without HTP were digested in the one phase and two-phase anaerobic digesters, respectively. Although the specific biogas production of MSWs in two-phase anaerobic digestion was significantly enhanced by HTP, no benefit could be found in one-phase anaerobic digestion because of the melanoidins formation. The specific biogas production of hydrothermally pretreated MSWs was much higher in two-phase anaerobic digestion (0.71Lbiogas/gVSadded) than that of MSWs in one-phase anaerobic digestion with or without HTP (0.53L and 0.55Lbiogas/gVSadded) when the butyric acid type fermentation dominated in the acidogenic phase. These findings indicated that the recalcitrant melanoidins formed in the HTP process were probably removed in the acidogenic digester. Differing from previous study, mixed-acid type fermentation, rather than ethanol type fermentation, was observed in the acidogenic digester fed with hydrothermally pretreated feedstock when pH was adjusted in the range of 4.0–4.5 with high oxidation reduction potential values of 49–97mV. The succeeding specific biogas production of mixed-acid type feedstock was only 0.59Lbiogas/gVSadded, which was lower than that of the butyric acid type feedstock. Microbial community structure was independent of the pH values and was substrate-specific in the acidogenic digesters. In the acidogenic digester with the HTP substrate, the phylum Firmicutes and Bacteroidetes increased, thereby possibly resulting from the increased content of soluble sugar and proteins in the HTP feedstock. Energy balance estimation of the four defined scenarios indicated that the combined HTP and two-phase AD in the MSWs treatment can achieve higher net energy output by 50.5–97.4% compared to the other three scenarios. This enables the combined process of HTP and two-phase AD to be a promising alternative way in the treatment of feedstock rich in protein and sugar.

Comparison of micro-aerobic and anaerobic fermentative hydrogen production from corn straw

Micro-aerobic and anaerobic fermentative hydrogen productions were carried out in bottle tests (150 mL) and scale-up bioreactors (10 L) using corn straw as a substrate. Hydrogen yield of 41.6 mL/g (volatile solid, VS) obtained from scale-up experiment with optimal oxygen addition of 0.28 mL/(g (total solid, TS)·day) was higher than that of 28.9 mL/g VS without additional oxygen. Oxidation reduction potential was−300∼−150 mV for micro-aerobic fermentation, where it was−560∼−360 mV for anaerobic fermentation. The micro-aerobic condition had strong buffering ability and could tolerate a greater accumulation of volatile fatty acid. Both micro-aerobic and anaerobic hydrogen production were butyric acid-type fermentation. Microbial community analysis indicated the presence of Citrobacter sp. and Escherichia sp., which were unique to micro-aerobic system, as well as classical Enterococcus, Bacteroides, Peptoclostridium, Clostridium genera that existed in both micro-aerobic and anaerobic fermentation.

Enhancement of Acidogenic Fermentation of Corn Stover Hydrolysates by Thermal Pretreatment with Diluted Formic Acid as Catalyst

In this study, formic acid was selected as the catalyst to pretreat corn stover which is also a good substrate for biohydrogen and biogas production. The pretreatment effect and the acidogenic characteristic of hydrolysates were also evaluated. Using corn stover (8.00 g) mixed with 72.00 g of formic acid (2.5%) at temperature 190 °C for reaction time 10 min, 17.36 ± 0.80 g/L glucose, 18.13 ± 0.83 g/L xylose, 5.45 ± 0.25 g/L arabinose, 3.88 ± 0.17 g/L acetic acid, and 1.96 ± 0.07 g/L furfural were obtained in the hydrolysate. The kinetic parameters of the Saeman model were determined to predict the percentage of xylan remaining in the substrate and the xylose in the liquid. Two kinds of specific hydrolysates (the highest yield of total sugar and highest yield of furfural) in the following acidogenic fermentation experiments were examined. The results indicated that both of the hydrolysates were shown to be butyric acid type fermentation and got the highest VFAs concentration of 6.3 ± 0.48 g/L. Diluted form...

Improving biomethane yield by controlling fermentation type of acidogenic phase in two-phase anaerobic co-digestion of food waste and rice straw

Controlling conditions to obtain different acidogenic fermentation types in a two-phase anaerobic co-digestion were investigated for improving the bio-methane yield from food waste (FW) and rice straw (RS). The results showed that the fermentation type in the acidogenic phase could be transformed by changing oxidation–reduction potential (ORP), pH, and organic loading rate (OLR). Three kinds of fermentation types were observed in acidogenic phase. Ethanol-type fermentation occurred at pH<4.5 and ORP<−120mV. Butyric acid-type fermentation appeared in the condition of pH>5.0 with lower ORP and OLR⩽6gVS/Ld. Mixed type fermentation was found in the transformation between butyric acid-type fermentation and ethanol-type fermentation. Among three fermentation types, the butyric-type fermentation achieved the highest acidification degree of 36%, biomethane yield of 535mLCH4/gVS, and biodegradability of 95%. The finding indicated that controlling fermentation type of acidogenic phase could be one of effective method for achieving high biomethane production.

Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter

The use of kissiris as promoter (culture immobilization carrier) in anaerobic acidogenesis of sucrose, raffinose and vinasse is reported. Initially, the effect of pH (4–8) and fermentation temperature (18–52°C) on the accumulation of low molecular weight organic acids (OAs) during sucrose acidogenesis, was evaluated. The promoting effect of kissiris was confirmed compared to free cells, resulting in 80% increased OAs production. The optimum conditions (pH 8; 37°C) were used during acidogenesis of sucrose/raffinose mixtures. A continuous system was also operated for more than 2months. When sucrose and sucrose/raffinose mixtures were used, lactic acid type fermentation prevailed, while when vinasse was used, butyric acid type fermentation occurred. Total OAs concentrations were more than 14g/L and ethanol concentrations were 0.5–1mL/L. Culture adaptation in vinasse was necessary to avoid poor results. The proposed process is promising for new generation ester-based biofuel production from industrial wastes.

Integration of fermentative biohydrogen with methanogenesis from fruit–vegetable waste using different pre-treatments

Fruit–vegetable waste was subjected to three different pre-treatments to enrich the two-stage biofuel production potential. The fluorescence excitation–emission matrix (EEM) spectra coupled with parallel factor (PARAFAC) analysis and fluorescence regional integration analysis were utilised to investigate dissolved organic matter degradation during two-stage fermentation process. The results showed that compared with that of alkali and enzyme pre-treatments, the acid pre-treatment resulted in the maximum biogas production rates and proportion in the hydrogenogenic stage (10.11mL/h, 41.2% hydrogen) when combined with the methanogenic process (4.67mL/h, 76.1% methane). In addition, the analysis of soluble metabolites composition indicated that both ethanol- and butyric acid-type fermentation processes had taken place as a result of acid pre-treatment, whereas only butyric acid-type fermentation resulted from alkali and enzyme pre-treatments. The PARAFAC analysis modelling of the EEM spectra revealed three fluorescent components in the effluents of three fermentation stages and assumed that the projected characteristic value may be used as a rapidly obtained indicator for substrate degradation and system stability of a two-stage biofuel production process.

Pretreatment of corn stover with diluted acetic acid for enhancement of acidogenic fermentation

A Box-Behnken design of response surface method was used to optimize acetic acid-catalyzed hydrothermal pretreatment of corn stover, in respect to acid concentration (0.05-0.25%), treatment time (5-15 min) and reaction temperature (180-210°C). Acidogenic fermentations with different initial pH and hydrolyzates were also measured to evaluate the optimal pretreatment conditions for maximizing acid production. The results showed that pretreatment with 0.25% acetic acid at 191°C for 7.74 min was found to be the most optimal condition for pretreatment of corn stover under which the production of acids can reach the highest level. Acidogenic fermentation with the hydrolyzate of pretreatment at the optimal condition at the initial pH=5 was shown to be butyric acid type fermentation, producing 21.84 g acetic acid, 7.246 g propionic acid, 9.170 butyric acid and 1.035 g isovaleric acid from 100g of corn stover in 900 g of water containing 2.25 g acetic acid.

Effect of Substrate Concentration on Hydrogen Production from Fermentation of Sugar Wastewater

The biological hydrogen production by the sugar wastewater is an effective way to achieve the reclamation. In this paper, the effect of substrate concentration on the hydrogen production is discussed through employing the self-made continuous flow anaerobic fermentation hydrogen production reactor, taking the sludge in urban sewage treatment plant as the inoculated sludge and the simulated sugar wastewater as the substrate. The experimental results show that the best hydrogen production effect can be obtained when the temperature is (37±1) °C, HRT is 7h, the water alkalinity is around 530mg/L and the substrate concentration is 5000mg/L, namely the organic load is 60kgCOD/(m3·d). The volumes of gas production and hydrogen production both reach the maximum. The average values are respectively 36.2L/d and 21.8L/d. The obtained hydrogen production rate is 0.93kgCOD/(m3·d). During the whole process, the proportion of volatile acid composition remains stable, which is the butyric acid-type fermentation. When the concentration of COD is increased to 6000-8000mg/L, the ability of hydrogen production of system will be significantly dropped due to the increase of pH of system. The hydrogen production performance can be restored through artificially and timely lowering the water alkalinity. However, the hydrogen production rate will be decreased compared to the previous situation.