Assessing optimal fermentation type for bio-hydrogen production in continuous-flow acidogenic reactors

In this study, the optimal fermentation type and the operating conditions of anaerobic process in continuous-flow acidogenic reactors was investigated for the maximization of bio-hydrogen production using mixed cultures. Butyric acid type fermentation occurred at pH>6, propionic acid type fermentation occurred at pH about 5.5 with E(h) (redox potential) >-278mV, and ethanol-type fermentation occurred at pH<4.5. The representative strains of these fermentations were Clostridium sp., Propionibacterium sp. and Bacteriodes sp., respectively. Ethanol fermentation was optimal type by comparing the operating stabilities and hydrogen production capacities between the fermentation types, which remained stable when the organic loading rate (OLR) reached the highest OLR at 86.1kgCOD/m(3)d. The maximum hydrogen production reached up to 14.99L/d.     

Effect of pH on phase separated anaerobic digestion

A pilot scale experiment was performed for a year to develop a two-phase anaerobic process for piggery wastewater treatment (COD: 6,000 mg/L, BOD: 4,000 mg/L, SS: 500 gm/L, pH 8.4, alkalinity 6,000 mg/L). The acidogenic reactor had a total volume of 3 m³, and the methanogenic reactor, an, anaerobic up-flow sludge filter, combining a filter and a sludge bed, was also of total volume 3 m³ (1.5 m³ of upper packing material). Temperatures of the acidogenic and methanogenic reactors kept at 20°C and 35°C., respectively. When the pH of the acidogenic reactor was controlled at 6.0–7.0 with HCl, the COD removal efficiency increased from 50 to 80% over a period of six months, and as a result, the COD of the final effluent fell in the range of 1,000–1,500 mg/L. BOD removal efficiency over the same period was above 90%, and 300 to 400 mg/L was maintained in the final effluent. The average SS in the final effluent was 270 mg/L. The methane production was 0.32 m³ CH₄/kg COD_removed and methane content of the methanogenic reactor was high value at 80–90%., When the pH of the acidogenic reactor was not controlled over the final two months, the pH reached 8.2 and acid conversion decreased compared with that of pH controlled, while COD removal was similar to the pH controlled operation. Without pH control, the methane content in the gas from methanogenic reactor improved to 90%, compared to 80% with pH control.     

Study of the acidogenic phase of the anaerobic fermentation of stillage from ethanol distilleries

Summary A bench scale continuously stirred reactor was used to study the acidogenic phase of the anaerobic fermentation of stillage. The residence time of the effluent in the reactor ranged from 15.7 to 8.2 hours, pH was around 5.0 and temperature was maintained at 35°C. The results indicate that the residence time had no appreciable effect on the production or composition of the organic acids. The main acid products found in the reactor effluent were acetic, propionic and butiric acids.     

Production of PHA from starchy wastewater via organic acids

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.     

Response of a biohydrogen-producing reactor to the substrate shift from sucrose to lactose

The response of an upflow acidogenic granule-based reactor to the substrate shift from sucrose to lactose was investigated in this study. Experimental results show that it took 60h for the reactor to completely degrade the new substrate. Hydrogen production performance, in terms of H(2) partial pressure, H(2) production rate and H(2) yield, was affected. Acetate, propionate, butyrate, valerate, caporate, ethanol and propanol were present in the reactor effluent, and their distribution changed significantly after the substrate shift. As the substrate was changed, the caproate- and ethanol-type fermentation was weakened, while the propionate-type fermentation was strengthened. Throughout the experiment, the butyrate-type fermentation played an important role. The H(2) yield had a close correlation with both propionate and B/A (butyrate/acetate) in this substrate shift process.     

Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses

A three-stage process was developed to produce polyhydroxyalkanoates (PHAs) from sugar cane molasses. The process includes (1) molasses acidogenic fermentation, (2) selection of PHA-accumulating cultures, (3) PHA batch accumulation using the enriched sludge and fermented molasses. In the fermentation step, the effect of pH (5–7) on the organic acids profile and productivity was evaluated. At higher pH, acetic and propionic acids were the main products, while lower pH favoured the production of butyric and valeric acids. PHA accumulation using fermented molasses was evaluated with two cultures selected either with acetate or fermented molasses. The effect of organic acids distribution on polymer composition and yield was evaluated with the acetate selected culture. Storage yields varied from 0.37 to 0.50 Cmmol HA/Cmmol VFA. A direct relationship between the type of organic acids used and the polymers composition was observed. Low ammonia concentration (0.1 Nmmol/l) in the fermented molasses stimulated PHA storage (0.62 Cmmol HA/Cmmol VFA). In addition, strategies of reactor operation to select a PHA-accumulating culture on fermented molasses were developed. The combination of low organic loading with high ammonia concentration selected a culture with a stable storage capacity and with a storage yield (0.59 Cmmol HA/Cmmol VFA) similar to that of the acetate-selected culture.     

Hydrogen-producing capability of anaerobic activated sludge in three types of fermentations in a continuous stirred-tank reactor

A continuous stirred-tank reactor was used as an anaerobic sludge system and the hydrogen production capabilities of three typical fermentations, in terms of specific hydrogen production rates, were investigated under the same hydraulic retention times (8 h) and influent chemical oxygen demand (5000 mg/L) at 35 °C. The reactor was continuously fed with diluted molasses, while the pH and oxidation reduction potential in the reactor were regulated to control the type of fermentation. The specific hydrogen production rate of the anaerobic sludge reached 2.96 mol/kg mixed liquid volatile suspended solid (MLVSS)/day, (mol•kg MLVSS^− 1 d^− 1), in ethanol-type fermentation, while 0.57 mol·kg MLVSS^− 1 d^− 1 in butyric acid-type fermentation, and 0.022 mol·kgMLVSS^− 1 d^− 1 in propionic acid-type fermentation. The hydrogen production capability of ethanol-type fermentation was 4.11 times greater than that of butyric acid-type fermentation and 148 times that of propionic acid-type fermentation.     

Anaerobic membrane reactor with phase separation for the treatment of cheese whey

Two-phase anaerobic digestion of cheese whey was investigated in a system consisting of a stirred acidogenic reactor followed by a stirred methanogenic reactor, the latter being coupled to a membrane filtration system to enable removal of soluble effluent whilst retaining solids. The acidogenic reactor was operated at a hydraulic retention time (HRT) of one day, giving maximum acidification of 52.25% with up to 5 g/l volatile fatty acids, of which 63.7% was acetic acid and 24.7% was propionic acid. The methanogenic reactor received an organic load up to 19.78 g COD/ld, corresponding to a HRT of 4 days, at which 79% CODs and 83% BOD(5) removal efficiencies were obtained. Average removals of COD, BOD(5) and TSS in the two-phase anaerobic digestion process were 98.5%, 99% and 100%, respectively. The daily biogas production exceeded 10 times reactor volume and biogas methane content was greater than 70%.     

Effect of substrate loading rate of chemical wastewater on fermentative biohydrogen production in biofilm configured sequencing batch reactor

The influence of substrate loading rate on fermentative hydrogen (H2) production was studied in biofilm configured sequencing batch reactor using chemical wastewater as substrate. Reactor was operated with selectively enriched anaerobic mixed microflora at different organic loading rates (OLRs; 6.3, 7.1 and 7.9kg COD/m3 day) after adjusting the feed to a pH of 6.0 (acidophilic) to provide suitable environment for acidogenic bacterial function. Variation in H2 production rate was observed with change in OLR [specific hydrogen yield - 13.44molH2/kgCODRday (6.3kgCOD/m3day), 8.23molH2/kgCODRday (7.1kgCOD/m3 day) and 6.064molH2/kgCODR day (7.9kgCOD/m3 day)]. H2 yield showed reasonably good correlation with pH drop [6.3kgCOD/m3 day (R2 - 0.9796), 7.1kgCOD/m3 day (R2 - 0.9973), 7.9kgCOD/m3 day (R2 - 0.9908)]. Increase in OLR showed marked reduction in COD removal efficiency [22.6% - 6.3kgCOD/m3 day; 19.8% - 7.1kgCOD/m3 day and 17.2% - 7.9kgCOD/m3 day].     

Studies on reaction techniques concerning reactor design for the anaerobic degradation of complex substrates with the example of the methanation of effluents in the fermentation industry

Two fixed-bed loop reactors were used to evaluate singleand separated-phase anaerobic treatments of a high strength waste-water from ethanol fermentation. The one-phase system consisted of an anaerobic fixed-bed loop reactor containing both acidogenic as well as methanogenic populations allowing a complete conversion of the carbon source into gaseous end products and biomass.The two-phase system consisted of a second fixed-bed loop reactor operated as a methanogenic unit, which was proceeded by a CSTR for acidification, both connected in series allowing sequential acidogenesis and methanogenesis of the organic components. The reactors were operated under steady state and variable process conditions. By gradually increasing the feed supply in both systems, maximum turnover of COD was determined.The separated-phase system consistently gave a better quality effluent with lower suspended solids and total COD. Maximum loading rates and COD elimination of the methanogenic phase of the two-phase system was over two times higher than that of the one-phase system. Process stability was also higher.On overloading the methane reactor of the two phase system accumulation of different fatty acids within the reactor was observed. Hydrogen concentration in the biogas can be used as a reliable indicator for system overloadings. At least, continuous online monitoring of hydrogen in the methanogenic reactor gas should provide a convenient alternative to other analyses for process control.     

The performance of a phase separated granular bed bioreactor treating brewery wastewater

This study presents the performance characteristics of a plug flow phase separated anaerobic granular bed baffled reactor (GRABBR) fed with brewery wastewater at various operating conditions. The reactor achieved chemical oxygen demand (COD) removal of 93-96% with high methane production when operated at organic loading rates (OLRs) of 2.16-13.38kg COD m(-3)d(-1). The reactor configuration and microbial environment encouraged the acidogenic dominant zone to produce intermediate products suitable for degradation in the predominantly methanogenic zone. Noticeable phase separation between acidogenesis and methanogenesis mainly occurred at high OLR, involving a greater number of compartments to contribute to wastewater treatment. The highly active nature and good settling characteristics of methanogenic granular sludge offered high biomass retention and enhanced methanogenic activities within the system. The granular structure in the acidogenic dominant zone of the GRABBR was susceptible to disintegration and flotation. Methanogenic granular sludge was a multi-layered structure with Methanosaeta-like organisms dominant in the core.     

Effect of sulphidogenesis on acid-phase digestion of waste activated sludge

This paper reports the influence of sulfate-reducing bacterial activity on acidogenic digestion of waste activated sludge (WAS). A series of experiments was conducted by feeding WAS to a 10-l, completely mixed, mesophilic reactor maintained at pH 5.0 under sulfidogenic and non-sulfidogenic conditions. Analyses of volatile fatty acids indicated that the productions of acetic and propionic acids were slightly increased by sulfidogenic activity at 218 mg/l level of sulfate when COD sulfate ratio was 55:1. Higher amounts of methanol, ethanol and hydrogen were observed in the non-sulfidogenic condition, but ammonia was lower than in the moderate sulfidogenic runs. At 0.63 kg VS/m³ d loading rate the hydrolysis was above 90% in both moderate sulfidogenic and non- sulfidogenic runs. The results of these experiments showed the possible influence of moderate sulfidogenesis in the protein degradation of WAS anaerobic digestion. However, the acid-phase digestion was adversely affected by increasing the sulfate concentration to 400 mg/l.     

Effects of key operational parameters on biohydrogen production via anaerobic fermentation in a sequencing batch reactor

In this study, a series of tests were conducted in a 6 L anaerobic sequencing batch reactor (ASBR) to investigate the effect of pH, hydraulic retention time (HRT) and organic loading rate on biohydrogen production at 28 °C. Sucrose was used as the main substrate to mimic carbohydrate-rich wastewater and inoculum was prepared from anaerobic digested sludge without pretreatment. The reactor was operated initially with nitrogen sparging to form anaerobic condition. Results showed that methanogens were effectively suppressed. The optimum pH value would vary depending on the HRT. Maximum hydrogen production rate and yield of 3.04 L H₂/L reactor d and 2.16 mol H₂/mol hexose respectively were achieved at pH 4.5, HRT 30 h, and OLR 11.0 kg/m³ d. Two relationships involving the propionic acid/acetic acid ratio and ethanol/acetic acid ratio were derived from the analysis of the metabolites of fermentation. Ethanol/acetic acid ratio of 1.25 was found to be a threshold value for higher hydrogen production.     

Fermentative hydrogen production from fresh leachate in batch and continuous bioreactors

This research for the first time investigated hydrogen production from the fresh leachate originated from municipal solid wastes. We found that fermentation of the leachate generated H₂ and was very much enhanced in the presence of extra phosphate in the batch reactor. The continuous expanded granular sludge bed (EGSB) reactor started to generate H₂ at day 20 and continued to 176 days with 120 mg/l of extra phosphate present. The highest chemical oxygen demand (COD) removal efficiency (66.9%) was achieved at liquid up-flow velocity of 3.7 m/h and hydraulic retention time of 12 h. Under proposed optimal operation conditions, the mean H₂ production rate reached up to 2155 ml/(l day). We also found that over 80% liquid metabolites were acetic acid and ethanol, suggesting the ethanol-type fermentation was dominant in the bioreactor. These findings indicate that the fresh leachate can be used as the source for continuous hydrogen production.     

Hydrogen bio-production through anaerobic microorganism fermentation using kitchen wastes as substrate

In order to treat the kitchen wastes and produce hydrogen, anaerobic fermentation technology was used in this experiment. The results showed that the fermentation type changed from mixed acid fermentation to ethanol fermentation in a continuous stirred tank reactor (CSTR) 22 days after start-up. The maximum efficiency of hydrogen bio-production in the CSTR was 4.77 LH₂/(L reactor d) under the following conditions: organic loading rate (OLR) of 32–50 kg COD/(m³ d), oxidation reduction potential (ORP) of −450 to −400 mV, influent pH value of 5.0–6.0, effluent pH value of 4.0–4.5, influent alkalinity of 300–600 mg/l, temperature of 35 ± 1°C and hydraulic retention time (HRT) of 7 h. An artificial neural network (ANN) model was established, and each parameter influencing the performance of the reactor was compared using the method of partitioning connection weights (PCW). The results showed that OLR, pH, ORP and alkalinity could influence the fermentation characteristics and hydrogen yield of the anaerobic activated sludge; with an influence hierarchy: OLR > pH values > ORP > alkalinity. An economic analysis showed that the cost of producing hydrogen in this experiment was less than the cost of electrolysis of water.     

Anaerobic Hydrogen Production from Sucrose Using an Acid‐Enriched Sewage Sludge Microflora

A novel and high‐rate anaerobic sequencing bath reactor (ASBR) process was used to evaluate the hydrogen productivity of an acid‐enriched sewage sludge microflora at a temperature of 35 °C. In this ASBR process a 4 h cycle, including feed, reaction, settle, and decant steps, was repeatedly performed in a 5 L reactor. The sucrose substrate concentration was 20 g COD/L; the hydraulic retention time (HRT) was maintained at 12–120 h at the initial period and thereafter at 4–12 h. The reaction/settle period ratio, which is the most important parameter for ASBR operation was 1.7. The experimental results indicated that the hydrogenic activity of the sludge microflora was HRT‐dependent and that proper pH control was necessary for a stable operation of the bioreactor. The peak hydrogenic activity value was attained at an HRT of 8 h and an organic loading rate of 80 kg COD/m³ × day. Each mole of sucrose in the reactor produced 2.8 mol of hydrogen and each gram of biomass produced 39 mmol of hydrogen per day. An overly‐short HRT might deteriorate the hydrogen productivity. The concentration ratios of butyric acid to’acetic acid, as well as volatile fatty acid and soluble microbial products to alkalinity can be used as monitoring indicators for the hydrogenic bioreactor.     

Effects of pH and ORP on microbial ecology and kinetics for hydrogen production in continuously dark fermentation

The microbial structure and kinetic characteristics of the hydrogen producing strains in two fermentative continuous stirred-tank reactors (CSTRs) were studied by controlling pH and oxidation and reduction potential (ORP). The fluorescence in situ hybridization (FISH) tests were conducted to investigate the fermentative performance of Clostridium histolyticum (C. histolyticum), Clostridium lituseburense (C. lituseburense) and Enterobacteriaceae. The experimental results showed that in ethanol-type reactor 1#, the relative abundance of the strains was 48%, 30% and 22%. Comparatively, the relative abundance in butyric acid-type reactor 2# was 24%, 55% and 19% with butyric acids and hydrogen as the main products. The kinetic results indicated that the hydrogen yield coefficients Y_P/X in both reactors were 8.357 and 5.951 l-H₂/g, while the coefficients of the cellular yield were 0.0268 and 0.0350 g-Cell/g, respectively. At the same biomass, the hydrogen yield in ethanol-type reactors was more than that in butyric acid reactors. However, the cellular synthesis rate in ethanol-type reactors was low when the same carbon source was used.     

Performance of a glucose fed periodic anaerobic baffled reactor under increasing organic loading conditions: 1. Experimental results

The influence of the organic loading rate on the performance of an innovative reactor, the periodic anaerobic baffled reactor (PABR) was examined. A laboratory-scale PABR of four compartments being fed with a glucose based synthetic medium performed with high stability while the feed organic load was doubled from 12.5 to 25 and then to 50 gCOD/l. Finally the feed concentration was increased to 75 gCOD/l. The successive step changes in the feed concentration lasted for 20, 15, and 7 d, respectively. The COD removal efficiency of the PABR was satisfactory in the first two transitions (approximately 97.5 and 96%). In the third transition (OLR=18.75 gCOD/l/d) the reactor failed as the pH dropped to 4. The concentrations of butyric and valeric acids increased as the organic loading was increased and eventually they became greater than the concentration of acetic and propionic acids.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang