Thermophilic hydrogen production from starch wastewater using two-phase sequencing batch fermentation coupled with UASB methanogenic effluent recycling

The aim of this study was to evaluate the performance of thermophilic hydrogenesis coupled with mesophilic methanogenesis in which the effluent was recycled to the hydrogen reactor for starch wastewater treatment. With this system, the hydrogen production rate and yield were 3.45 ± 0.25 L H₂/(L·d) and 5.79 ± 0.41 mmol H₂/g COD_added respectively, and thus higher than the values of the control group without methanogenic effluent recycling. In addition, relatively higher contents of acetate and butyrate were obtained in the hydrogen reactor with recirculation. The methane reactors were operated with the effluent from the hydrogen reactor, and methane yield was stabilized at 0.21–0.23 L/g COD_removal in both. Analysis of the microbial communities further showed that methanogenic effluent recirculation enriched microbial communities in the hydrogen reactor. Two species of bacteria effective in hydrogenesis, Thermoanaerobacterium thermosaccharolyticum and Clostridium thermosaccharolyticum, dominated during hydrogen production, whereas archaea belonging to Euryarchaeota were detected and cultured in the methane reactor. The recycled effluent supplied alkaline substrates for the hydrogen producing bacteria. Alkali balance calculations showed that the amount of added alkali was reduced by 88%. This amount, required for hydrogen production from starch wastewater, was contributed by alkali in the methanogenic effluent, (2225 ± 140 mg CaCO₃/L), resulting in lower operational costs.     

Hydrogen and methane potential based on the nature of food waste materials in a two-stage thermophilic fermentation process

The purpose of this study is to investigate the biological H₂ and CH₄ potential based on the nature of organic waste materials in a two-stage thermophilic fermentation process. Three varieties of actual waste specifically potato, kitchen garbage and bean curd manufacturing waste (okara) were selected. The production rates for H₂ and CH₄ were as follows: potato, 2.1 and 1.2 l/l/d; garbage, 1.7 and 1.5 l/l/d; okara, 0.4 and 1.4 l/l/d in the continuous processes. The H₂ and CH₄ yields were 20–85 ml H₂/g VS_added and 329–364 ml CH₄/g VS_added, respectively. The H₂ yield increased and the CH₄ yield decreased in the order of potato, kitchen garbage and okara. The H₂ yield was shown to be not only dependent on the proportion of carbohydrate but also on the hydrolysis pH of the organic waste, which was influenced by the nature of the organic waste materials. Higher yields of H₂ or CH₄ were obtained when the hydrolysis pH of the organic waste was close to the optimum pH range of H₂-producing bacteria or methanogenic archaea in the two-stage fermentation processes.     

An online-monitored thermophilic hydrogen production UASB reactor for long-term stable operation

In this study, the H₂ production and chemical oxygen demand (COD) removal performances of a thermophilic upflow anaerobic sludge blanket (UASB) reactor with online monitoring system were investigated. The online monitoring system enabled a rapid monitoring and timely control of the process. As a consequence, high operating stability was achieved despite of the varied hydraulic retention time (HRT) during 310-day operation. The COD efficiency remained at above 98%, and the hydrogen yield fluctuated slightly within the range of 2.42–3.06 mol H₂ mol⁻¹ sucrose. Thermophilic H₂-producing granules were successfully cultivated in this reactor, which showed better physical and microbial properties than floc sludge and higher H₂ production rate than mesophilic granules. An analysis of the microbial growth kinetics further demonstrated a possibly higher synthesis and metabolism activity of microbes in the thermophilic granule state.     

Natural inducement of hydrogen from food waste by temperature control

An easy and simple method of producing H₂ from food waste was devised. Although there was no inoculum addition or pretreatment, food waste was naturally decomposed and converted to H₂ when cultivated at 50-60 °C in anaerobic state. Both the highest H₂ yield of 1.79 mol H₂/mol hexose_added and a production rate of 369.1 ml H₂/L/h were observed at 50 °C. While butyrate was the main by-product of the food waste cultivated at 50 °C, lactate whose producing-reaction is non-hydrogenic was dominant at 35 °C where the worst performance was observed. The degradation efficiency of volatile solids and carbohydrate was similar to 50% and 90%, respectively, at both temperatures. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis clearly revealed that the role of temperature control was the microbial selection. At high temperature, the activity of indigenous lactic acid bacteria was suppressed while H₂-producing bacteria, such as Clostridium sp., Acetanaerobacterium elongatum, and Caloramater indicus, were predominantly cultivated.     

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.     

Inoculum pretreatment differentially affects the active microbial community performing mesophilic and thermophilic dark fermentation of xylose

The influence of different inoculum pretreatments (pH and temperature shocks) on mesophilic (37 °C) and thermophilic (55 °C) dark fermentative H₂ production from xylose (50 mM) and, for the first time, on the composition of the active microbial community was evaluated. At 37 °C, an acidic shock (pH 3, 24 h) resulted in the highest yield of 0.8 mol H₂ mol^?1 xylose. The H₂ and butyrate yield correlated with the relative abundance of Clostridiaceae in the mesophilic active microbial community, whereas Lactobacillaceae were the most abundant non-hydrogenic competitors according to RNA-based analysis. At 55 °C, Clostridium and Thermoanaerobacterium were linked to H₂ production, but only an alkaline shock (pH 10, 24 h) repressed lactate production, resulting in the highest yield of 1.2 mol H₂ mol^?1 xylose. This study showed that pretreatments differentially affect the structure and productivity of the active mesophilic and thermophilic microbial community developed from an inoculum.     

Long term impact of stressing agents on fermentative hydrogen production: Effect on the hydrogenase flux and population diversity

In this study, the long term effect of different microbial stressing agents on hydrogen (H₂) production was examined using repeated batch cultivations. When compared to thermophilic cultures, higher H₂ yields were observed in mesophilic cultures receiving repeated glucose addition. Methane production was only observed in control mesophilic cultures receiving repeated 5 glucose additions. Lower hydrogenase evolution specific activity was observed in thermophilic cultures (except alkali-treated cultures) compared to mesophilic cultures. For both mesophilic and thermophilic cultures, the hydrogenase uptake specific activity of the untreated control cultures exhibited higher levels of activity than the pretreated cultures. A flux balance analysis (FBA) showed negligible homoacetogenic flux in mesophilic cultures pretreated with linoleic acid (LA) and loading shock (LS) after successive batch cultivations. The homoacetogenic flux accounted for approximately 98% loss in the H₂ yield in untreated mesophilic control cultures. Both homoacetogens (Eubacterium sp.) and aceticlastic methanogens (Methanosaeta sp. and Methanosarcina sp.) were abundant in the control cultures. In comparison, Clostridium sp. were dominant in mesophilic stress treated cultures whereas under thermophilic conditions, the dominant microorganisms were Flavobacterium sp., Bacillus sp., Thermoanaerobacter sp., Bacteroides sp., Lactobacillus sp. and Thioalkalivibrio sp.     

Effect of acid-pretreatment on hydrogen fermentation of food waste: Microbial community analysis by next generation sequencing

This work presents the effect of acid-pretreatment on H₂ fermentation of food waste with detailed microbial information by next generation sequencing. The pretreated food waste at pH 1.0–4.0 was cultivated under mesophilic conditions without external inoculum addition. From the food waste acid-pretreated at pH 1–3, H₂ yields in the range of 1.37–1.74 mol H₂/mol hexose_added were achieved, attaining the highest value at pH 2. Clostridium sp. such as Clostridium acetobutylicum ATCC 824 and Clostridium perfringens occupied more than 70% of total number of sequences at pH 1–3. On the other hand, in the control (no pretreatment) and at pH 4, lactic acid bacteria such as Lactobacillus and Streptococcus were found to be the dominant genus (>90% of total number of sequences), resulting in a low H₂ yield. In addition, the effect of substrate concentration on H₂ fermentation was investigated, and the maximum H₂ productivity was estimated to be 27.2 L H₂/L/d by Andrew's model.     

Hydrogen production from alcohol wastewater by an anaerobic sequencing batch reactor under thermophilic operation: Nitrogen and phosphorous uptakes and transformation

The objective of this study was to investigate hydrogen production from alcohol wastewater using an anaerobic sequencing batch reactor (ASBR) under thermophilic operation and at a constant pH of 5.5. Under the optimum COD loading rate of 68 kg/m³d, the produced gas contained 43% H₂ without methane and the system provided a hydrogen yield and specific hydrogen production rate of 130 ml H₂/g COD removed and 2100 ml H₂/l d, respectively, which were much higher than those obtained under the mesophilic operation. Under thermophilic operation, both nitrogen and phosphate uptakes were minimal at the optimum COD loading rate for hydrogen production and most nitrogen uptake was derived from organic nitrogen. Under the thermophilic operation for hydrogen production, the nutrient requirement in terms of COD:N:P was found to be 100:6:0.5, which was much higher than that for the methenogenic step for methane production under both thermophilic and mesophilic operations and for the acidogenic step for hydrogen production under mesophilic operation.     

Fungal solid-state fermentation of food waste for biohydrogen production by dark fermentation

The dark fermentation process was evaluated for biohydrogen production from food waste through fungal solid-state fermentation (SSF). Three fungal cultures (one strain of Aspergillus tubingensis and two strains of Meyerozyma caribbica) were compared, being A. tubingensis the best hydrolyser culture for releasing soluble carbohydrates. The biochemical hydrogen potential of food waste hydrolysate (FWH) at different substrate-inoculum ratios obtained a lower hydrogen yield than untreated food waste (RFW). The highest hydrogen yield value corresponded to treatments RFW-20 and RFW-30 with 77.0 ± 2.6 and 76.9 ± 1.4 mL H₂ normalized by per gram volatile solid added (NmL H₂/gVS_added), respectively. The microbial community of food waste was analysed, being detected lactic-acid bacteria genera as Latilactobacillus and Leuconostoc. The presence of actively growing bacteria during the SSF could explain the lowest hydrogen yield (20.1–36.0 NmL H₂/gVS_added) in the FWH treatment due to the substrate competition between lactic-acid bacteria and hydrogen-producing bacteria, where the lactic-acid bacteria were favoured by their faster growth rate.     

Two-stage UASB reactor converting coffee drink manufacturing wastewater to hydrogen and methane

In this study, the feasibility of a continuous two-stage up-flow anaerobic sludge blanket (UASB) reactor system, consisted of thermophilic (55 °C) dark fermentative H₂ production and mesophilic (35 °C) CH₄ production from coffee drink manufacturing wastewater (CDMW), was tested. A recently proposed operational strategy was used to overcome a major drawback of the long start-up period of the UASB reactor. Firstly, a completely stirred tank reactor (CSTR) was operated for 8 days to prepare seeding. The seed was then directly transferred to the UASB reactor. Microbial aggregation took place in the initial period, and the floc size was gradually increased over time. In UASB reactor, the maximum H₂ yield of 2.57 mol H₂/mol hexose_added and a stable H₂ production rate of 4.24 L H₂/L/h were observed at a hydraulic retention time (HRT) of 6 h and substrate concentration of 20 g Carbo. COD/L. In this novel method using CDMW, thermophilic H₂-producing granules with an average particle size of 1.3 mm was successfully developed after 100 days. The more bioenergy recovery was attempted in a post-treatment process using a mesophilic UASB reactor for CH₄ production from the H₂ fermented effluent. The maximum CH₄ yield of 325 mL of CH₄/g COD was achieved with removing 93% of the COD at an organic loading rate of 3.5 g COD/L/d. The developed two-stage UASB reactor system achieved biogas conversion by 88.2% (H₂ 15.2% and CH₄ 73%) and COD removal by 98%.     

Bacterial community analyses by pyrosequencing in dark fermentative H2-producing reactor using organic wastes as a feedstock

Hydrogen(H₂)-producing bacterial community structures of the dark fermentation system in a batch reactor were investigated during 48 h by analyzing 16S rRNA gene sequences obtained from pyrosequencing. Organic wastes composed of food waste and sewage sludge were used as a feedstock. After heat treatment (90 °C for 20 min) of the feedstock, H₂ was naturally evolved under anaerobic mesophilic conditions, showing a H₂ yield of 2.26 mol H₂/mol hexose_added. The bacterial community structure of the initial inoculum (microbial community at the starting point (0 h)) combined with heat treated food waste and sewage sludge was mainly comprised of Proteobacteria and Bacteroidetes. After 6 h operation, the sequences that belong to other groups except Firmicutes decreased dramatically and were not observed at all in the latter samples. Clostridium spp., which were negligible in the inoculum, took over the main bacterial community by taking charge of H₂ production. Among the phylum Firmicutes, the sequences closely related with Clostridium sordellii ATCC 9714^T, Clostridium perfringens ATCC 13124^T, and Clostridium butyricum ATCC 19398^T became predominant in the time series within 48 h. Overall, the results showed how fast the Clostridium spp. overwhelmed the bacterial community in dark fermentative H₂ production conditions, where they were at a negligible amount at the start.     

Effect of organic loading rate on continuous hydrogen production from food waste in submerged anaerobic membrane bioreactor

The characteristics of hydrogen fermentation in a membrane bioreactor (HF-MBR) fed with food waste were investigated at thermophilic condition. The HF-MBR was operated at three different organic loading rates (OLRs) of 70.2, 89.4 and 125.4 kg-COD/m³/day. Biogas production rate increased from 22.4 to 32.8 and 62.5 l/day with OLR. The maximum Hydrogen yield and production rate were 111.1 mL-H₂/g-VS _added and 10.7 l-H₂/L/day at an OLR of 125.4 kg-COD/m³/day. The total carbohydrate degradation was better than 96% throughout the experimental runs. Continuous H₂ production from food waste with CH₄-free biogas was successfully sustained in the HF-MBR for 90 days. The microbial community was dominated by Clostridium sp. strain Z6. The H₂ production was significantly improved by shortening the retention time and increasing the OLRs. The HF-MBR showed an H₂ production capacity at the high OLRs due to its higher cell retention.     

Evaluation of sludge liquors from acidogenic fermentation and thermal hydrolysis process as feedstock for microbial electrolysis cells

In this study, mesophilic acidogenic fermentation, thermophilic acidogenic fermentation, and thermal hydrolysis process (THP) were compared to generate sludge liquors for bioenergy recovery with microbial electrolysis cells (MECs). The results showed that THP at 170 °C was the most effective for hydrolysis of particulate organics in sewage sludge, while fermentation under thermophilic temperature led to the highest accumulation of volatile fatty acids (VFAs) in sludge liquor. However, THP yielded the highest percentage of acetate in VFAs, which resulted in superior MEC performance compared to fermented sludge liquors in terms of current density (2.7 vs. ~1.3 A/m²), coulombic efficiency (50% vs. 31–34%), bio-H₂ potential (1114 vs. 839–881 mL), and H₂ production rate (50.3 mL/d vs. 28–32 mL/d). The utilization sequence of the VFAs was found to be acetate > butyrate > propionate. Overall, our results show that generating sludge liquors through THP could provide a feasible solution to produce bio-H₂ from sewage sludge; however, coulombic efficiencies should be further improved before practical application.     

Hydrogen production performance from food waste using piggery anaerobic digested residues inoculum in long-term systems

The objective of this study was to investigate the hydrogen production performance from food waste using piggery anaerobic digested residues (PADRs) inoculum. Multiple parameters were evaluated such as organic load rate (OLR), pH, and hydraulic retention time (HRT), over a wide range of values in long-term dark fermentation systems. Results showed that a value of 126.50 mL/gVS·d hydrogen yield was achieved at OLR 6 g VS/L·d under thermophilic condition. A relatively stable structural composition dominated by Thermoanaerobacterium was maintained even suffering from OLR and acid shock. On the contrary, mesophilic fermentation performed acetic acids accumulation and an average hydrogen yield of less than 80 mL/gVS·d. High OLR and low pH (range of 5.0–5.5) led to the establishment of Lactobacillus. Beyond this range, the relative abundance of Olsenella, Streptococcus, and other bacteria showed a significant difference under different operating conditions, which caused weak resistance to external shocks during mesophilic fermentation. It showed that PADRs was capable of obtaining optimal hydrogen production performance under thermophilic condition from food waste with a stable microbial community structure.     

Thermophilic versus mesophilic dark fermentation in xylose-fed fluidised bed reactors: Biohydrogen production and active microbial community

Dark fermentative biohydrogen production in a thermophilic, xylose-fed (50 mM) fluidised bed reactor (FBR) was evaluated in the temperature range 55–70 °C with 5-degree increments and compared with a mesophilic FBR operated constantly at 37 °C. A significantly higher (p = 0.05) H₂ yield was obtained in the thermophilic FBR, which stabilised at about 1.2 mol H₂ mol^?1 xylose (36% of the theoretical maximum) at 55 and 70 °C, and at 0.8 mol H₂ mol^?1 xylose at 60 and 65 °C, compared to the mesophilic FBR (0.5 mol H₂ mol^?1 xylose). High-throughput sequencing of the reverse-transcribed 16S rRNA, done for the first time on biohydrogen producing reactors, indicated that Thermoanaerobacterium was the prevalent active microorganism in the thermophilic FBR, regardless of the operating temperature. The active microbial community in the mesophilic FBR was mainly composed of Clostridium and Ruminiclostridium at 37 °C. Thermophilic dark fermentation was shown to be suitable for treatment of high temperature, xylose-containing wastewaters, as it resulted in a higher energy output compared to the mesophilic counterpart.     

Trace metals supplementation enhanced microbiota and biohythane production by two-stage thermophilic fermentation

The effect of trace metals supplementation into palm oil mill effluent on biohythane production and responsible microbial communities in thermophilic two-stage anaerobic fermentation was investigated. High biohythane yields were linked to Ni/Co/Fe supplementation (10, 6 and 20 mg L⁻¹, respectively) with maximum H₂ and CH₄ yields of 139 mL H₂ gVS⁻¹ and 454 mL CH₄ gVS⁻¹, respectively. The Ni/Co/Fe supplementation resulted in higher numbers of Bacillus sp., Clostridium sp. and Thermoanaerobacterium sp. together with increasing hydrogenase expression level leading to increasing hydrogen yields of 90.4%. The numbers of Methanosarcina, Methanomassiliicoccus, and Methanoculleus were enhanced by Ni/Co/Fe addition, accompanied by 21.7% higher methane yields. No correlation between methyl coenzyme-M reductase expression level and methane yields was observed. The Ni/Co/Fe supplementation improved gas production in the two-stage biohythane process via enhancing a number of viable hydrogen-producing bacteria together with hydrogenase activity in H₂ stage and enhancing number methanogens in the CH₄ stage.     

Effects of inoculum and indigenous microflora on hydrogen production from the organic fraction of municipal solid waste

Biological production of hydrogen (H₂) by dark fermentation is an exciting scientific area for the conversion of low-cost residues and waste into biofuel. The main requirement for an efficient H₂ production process is the availability of efficient microbial consortia in which H₂-utilizing and non-H₂-producing bacteria are suppressed. This study was performed to evaluate the H₂ production potentials from the organic fraction of municipal solid waste (OFMSW) with and without addition of inoculum. The results showed that hydrogen productions from OFMSW without addition of inoculum were comparable to those obtained with inoculum but a latency phase of about 6 days occurred. On the contrary, addition of inoculum resulted in higher H₂ production potentials without any latency phase. The use of a properly pre-treated inoculum confirmed to be an interesting and improvable tool to obtain high H₂ yields from organic waste. However the indigenous OFMSW microbiota showed promising hydrogen yields especially toward the development of efficient hydrogen producing microbial inoculants.     

One-stage H2 and CH4 and two-stage H2 + CH4 production from grass silage and from solid and liquid fractions of NaOH pre-treated grass silage

In the present study, mesophilic CH₄ production from grass silage in a one-stage process was compared with the combined thermophilic H₂ and mesophilic CH₄ production in a two-stage process. In addition, solid and liquid fractions separated from NaOH pre-treated grass silage were also used as substrates. Results showed that higher CH₄ yield was obtained from grass silage in a two-stage process (467 ml g⁻¹ volatile solids (VS)_original) compared with a one-stage process (431 ml g⁻¹ VS_original). Similarly, CH₄ yield from solid fraction increased from 252 to 413 ml g⁻¹ VS_original whereas CH₄ yield from liquid fraction decreased from 82 to 60 ml g⁻¹ VS_original in a two-stage compared to a one-stage process. NaOH pre-treatment increased combined H₂ yield by 15% (from 5.54 to 6.46 ml g⁻¹ VS_original). In contrast, NaOH pre-treatment decreased the combined CH₄ yield by 23%. Compared to the energy value of CH₄ yield obtained, the energy value of H₂ yield remained low. According to this study, highest CH₄ yield (495 ml g⁻¹ VS_original) could be obtained, if grass silage was first pre-treated with NaOH, and the separated solid fraction was digested in a two-stage (thermophilic H₂ and mesophilic CH₄) process while the liquid fraction could be treated directly in a one-stage CH₄ process.     

Using a food and paper-cardboard waste blend as a novel feedstock for hydrogen production: Influence of key process parameters on microbial diversity

In this study, fermentation of a thermally treated simulated organic solid waste into hydrogen (H₂) was examined using a pretreated anaerobic mixed culture. The culture was fed a steam exploded food waste plus paper-cardboard waste blend liquor with and without linoleic acid (LA). The individual and interaction effects of the initial pH, LA concentration and the initial chemical oxygen demand (COD) concentration on H₂ and methane (CH₄) production was assessed using a Box–Behnken design (BBD). The BBD model predicted a maximum H₂ yield of 87 mL g⁻¹ COD or 98 mL H₂ g⁻¹ VS with 1.6 g L⁻¹ LA, an initial pH of 5.93 and an initial COD of 9.34 g COD L⁻¹. The major microbial populations detected in cultures at pH 5.5 with and without LA included Clostridium sp., Enterococcus asini, Enterococcus faecalis, and Lactobacillus gallinarum. The dendrogram for the 16S rRNA gene T-RFs profiles showed four major groups with a similarity index of 72–75% for Clade III. The major H₂-producing populations were grouped in Clade I with a similarity index range of 55–75%.