Development of a simple bio-hydrogen production system through dark fermentation by using unique microflora

In order to ensure efficient functioning of hydrogen fermentation systems that use Clostridium as the dominant hydrogen producer, energy-intensive process such as heat pretreatment of inoculum and/or substrate, continuous injection, and control of anaerobic conditions are required. Here, we describe a simple hydrogen fermentation system designed using microflora from leaf-litter cattle-waste compost. Hydrogen and volatile fatty acid production was measured at various hydraulic retention times, and bacterial genera were determined by PCR amplification and sequencing. Although hydrogen fermentation yield was approximately one-third of values reported in previous studies, this system requires no additional treatment and thus may be advantageous in terms of cost and operational control. Interestingly, Clostridium was absent from this system. Instead, Megasphaera elsdenii was the dominant hydrogen-producing bacterium, and lactic acid-producing bacteria (LAB) were prevalent. This study is the first to characterize M. elsdenii as a useful hydrogen producer in hydrogen fermentation systems. These results demonstrate that pretreatment is not necessary for stable hydrogen fermentation using food waste.     

Non-sterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis

Bio-hydrogen production from food waste by anaerobic mixed cultures was conducted in a continuous stirred tank reactor (CSTR). The hydraulic retention time (HRT) was optimized in order to maximize hydrogen yield (HY) and hydrogen production rate (HPR). The maximum hydrogen content (38.6%), HPR (379 mL H₂/L. d) and HY (261 mL H₂/g-VS_added) were achieved at the optimum HRT of 60 h. The major soluble metabolite products were butyric and acetic acids which indicated a butyrate-acetate type fermentation. Operation of CSTR at HRT 60 h could select hydrogen producing bacteria and eliminate lactic acid bacteria and acetogenic bacteria. The microbial community analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the predominant hydrogen producer was Clostridium sp.     

Microbial characterization of hydrogen-producing bacteria in fermented food waste at different pH values

An anaerobic fermentation of food waste was conducted in a 0.5 L bioreactor incubated at a thermophilic temperature of 55 °C to evaluate the effects of different controlled pH values (5.0, 5.5 and 6.0) on biohydrogen production. Effective biohydrogen production was found at controlled pH 5.5 and 6.0 corresponding to lower lactic acid production compared to pH 5.0. It was demonstrated that biohydrogen production from food waste was pH-dependent with hydrogen yields of 79, 76 and 23 mmol H₂/L-media/d for pH 5.5, 6.0 and 5.0, respectively. Specific microbial determination for Clostridium sp. and total bacteria quantification were carried out by the fluorescent in-situ hybridization (FISH) technique. The number of Clostridium sp. for acclimatized sludge, fermentation broth at pH 5.0, 5.5 and 6.0 were 2.9 × 10⁸, 3.6 × 10⁸, 7.8 × 10⁸ and 5.4 × 10⁸ cells/ml, respectively. The quantification analysis showed that 92% of the total bacteria belonged to Clostridium sp. from clusters I and XI from the sample at controlled pH 5.5. The denaturing gradient gel electrophoresis (DGGE) bands of the sample after heat-treatment, acclimatization and during fermentation indicated the presence of Bacteroidetes, Caloromator australicus sp. and Clostridium sp.     

Hydrogen production from acidogenic food waste fermentation using untreated inoculum: Effect of substrate concentrations

The effect of substrate concentrations (0, 7.5, 15, 22.5, 30, and 37.5 g-VS/L) on hydrogen production from heat-treated and fresh food waste (FW) using untreated inoculums was investigated in this work. The highest hydrogen yield (75.3 mL/g-VS) was obtained with heat-treated FW at 15 g-VS/L. Lower substrate content could not provide enough organic matter for hydrogen fermentation, while higher substrate concentrations shifted the metabolic pathways from hydrogen fermentation to lactic acid fermentation by enriching the lactic acid bacteria (LAB), which lowered the slurry pH and decreased enzyme activity, resulting in a lower chemical oxygen demand (COD), volatile solid (VS), carbohydrate removal rate, and hydrogen yield. Compared with fresh FW, heat-treated FW is preferred for biohydrogen process with acetate as the main organic product. Additionally, at the optimal concentration (15 g-VS/L) using fresh FW, lactic acid is first accumulated and then degraded to produce hydrogen with butyrate as the main metabolite.     

Accelerated startup of biological hydrogen production process by addition of Ethanoligenens harbinense B49 in a biofilm-based column reactor

Two biofilm-based column reactors with walnut shell (WS) as carrier media were applied for fermentative hydrogen production using glucose as substrate by mixed microbial cultures at the temperature of 35 °C. Pure hydrogen producing bacteria Ethanoligenens harbinense B49 was supplemented into the reacting system periodically or continuously to enhance hydrogen production ability at the startup period. The results showed that the bioreactors supplemented with E. harbinense B49 performed better than the reactor without bacteria addition. Continuous addition mode was recommended, since the hydrogen production performance was better and the operation was easily to be accomplished. The optimal addition amount of pure bacteria was also investigated. The optimal bacterial addition amount was found to be 2.5% which led a better hydrogen production rate. In addition, the bioreactor supplemented with pure bacteria continuously presented a high hydrogen production ability as the specific hydrogen production rate (SHPR) maximized at 1.36 L/g-VSS·d; whereas, the bioreactor without bacteria addition obtained a maximum specific hydrogen production rate of 1.10 L/g-VSS·d. The addition of E. harbinense B49 favored the transformation to ethanol type fermentation in the bioreactor. Thereby, the startup period had been accelerated remarkably.     

Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp.A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H₂/L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum-like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.     

Feasibility study on the application of rhizosphere microflora of rice for the biohydrogen production from wasted bread

We performed an experiment of continuous anaerobic hydrogen fermentation as a pilot-plant-scale test, in which waste from a bread factory was fermented by microflora of rice rhizosphere origin. The community structure of microflora during anaerobic hydrogen fermentation was analyzed using PCR-DGGE, FISH, and quinone profiles. The relation of those results to hydrogen generation was discussed. Results show that a suitable condition was a reactor temperature of 35 °C, with HRT 12–36 h, volume load of 30–70 kg-COD_Cr/m³ day, and maximum hydrogen production rate of 1.30 mol-H₂/mol-hexose. Regarding characteristics of microflora during fermentation, PCR-DGGE results show specific 16S rDNA band patterns; Megasphaera elsdenii and Clostridium sp. of the hydrogen-producing bacteria were identified. M. elsdenii was detected throughout the fermentation period, while Clostridium sp. of hydrogen-producing bacteria was detected on the 46th day. Furthermore, FISH revealed large amounts of Clostridium spp. in the sample. The quinone profile showed that the dominant molecular species of quinone is MK-7. Because Clostridium spp. belong to MK-7, results suggest that the quinone profile result agrees with the results of PCR-DGGE and FISH. Microflora in the rhizosphere of rice plants can be a possible resource for effective bacteria of biohydrogen production.     

Pilot-scale hydrogen fermentation system start-up performance

A high-rate hydrogen production process able to produce H₂ at a maximum rate of 15 L/L/h was successfully developed by the Feng Chia University (FCU) biohydrogen research team. This highly efficient hydrogen fermentation system includes a 400 L pilot-scale system constructed for determining scale-up operation parameters for commercializing the bioH₂ production technology. The pilot-scale system is composed of a feedstock tank, mixing system, fermentor, gas/liquid separator and automatic control system. The fermentor is fed with sucrose (20 g COD/L) and operated at 35 °C. A batch strategy is used for system start-up. The fermentor was first operated in a batch mode for two days and then switched to a continuous-feeding mode (HRT 12 h) for one month. During the continuous operation, pH notably affected H₂ production efficiency and bacterial community. For the first 14-day operation, the H₂ production rate increased from 0.017 to 0.256 L/L/h with a pH variation from 5.0 to 7.0. The DGGE results indicate the presence of two Clostridium species (namely, Clostridium butyricum and Clostridium pasteurianum) in the fermenter. Stable hydrogen production rate was obtained at pH 5.5–6.0 when C. pasteurianum became dominant in the mixed culture.     

Alkali-treated sewage sludge as a seeding source for hydrogen fermentation of food waste leachate

In the present work, alkali-treated sewage sludge (SS) was used as a seeding source for H₂ fermentation of food waste leachate (FWL). The role of alkaline treatment of SS was to suppress the activity of non-H₂-producers in SS and also to enhance the solubility of SS. The effect of pretreatment pH and FWL:SS ratio on H₂ production was crucial, by changing the pH conditions and selecting the dominant species. High pretreatment pH and high SS content resulted in high initial pH conditions. The highest H₂ yield of 2.1 mol H₂/mol hexose_added was achieved at pretreatment pH 10 and a mixing ratio of FWL:SS = 3:5. At these conditions, the initial pH was 7.9, and cultivation pH was maintained within the reported optimum range of 5.5–6.5 during fermentation. It was found that pretreatment pH 9 was not strong enough to suppress the activity of non-H₂-producers in SS, in particular, lactic acid bacteria (LAB). Microbial analysis results confirmed that LAB such as Lactobacillus sp. and Enterococcus sp. were the dominant species at pretreatment pH 9 while Clostridium sp., the main anaerobic H₂-producers, were dominant at pretreatment pH 10.     

Effect of extrinsic lactic acid on fermentative hydrogen production

In this paper we report the effect of extrinsic lactic acid on hydrogen production from a starch-containing medium by a mixed culture. Study of the effect of addition of four metabolites, namely ethanol, lactic acid, butyric acid and acetic acid illustrated that lactic acid had a positive effect on both the maximum hydrogen production and hydrogen production rate. The addition of 10 mM lactic acid to a batch containing starch increased the hydrogen production rate and hydrogen production yield from 4.31 to 8.23 mL/h and 5.70 to 9.08 mmol H₂/g starch, respectively. This enhancement in hydrogen production rate and yield was associated with a shift from acetic acid and ethanol formation to formation of butyric acid as the predominant metabolite. The increase in hydrogen production yield was attributed to the increase in the available residual NADH for hydrogen production. When lactic acid was used as the sole carbon source, no significant hydrogen production was observed.     

Acidophilic biohydrogen production from rice slurry

Batch experiment results showed that hydrogen production from rice slurry was found most effective at pH 4.5, 37 °C treating a slurry containing 5.5 g-carbohydrate/L. An anaerobic digester sludge was used as seed after a 100 °C heat treatment for 30 min. After a 36 h acclimation period, the sludge had a maximum specific hydrogen production rate of 2.1 L/(g-VSS d) and a hydrogen yield of 346 mL/g-carbohydrate, corresponding to 62.6% of stoichiometric yield. The effluent was composed mostly of acetate (28.3–43.0%) and butyrate (51.4–70.9%). Based on the 16S rDNA analysis, the 28 clones developed from this acidophilic hydrogen-producing sludge may be classified into nine OTUs, all of which are affiliated with the genus Clostridium. Phylogenetic analysis shows that eight OTUs (96.4% of population) form a distinct group with Clostridium sp. 44a-T5zd. Results indicate the acidophilic hydrogen-producing bacteria found in this study are unknown, and warrant further studies.     

Enzymatic routes to hydrogen and organic acids production from banana waste fermentation by autochthonous bacteria: Optimization of pH and temperature

The Central Composite Rotational Design (CCRD) was employed to find the optimum pH (5.09–7.91) and temperature (27.1–46,9 °C) for hydrogen production in banana waste (BW) fermentation by autochthonous microbial biomass. The P and Rm ranged between 6.06 and 62.43 mL H₂ and 1.13–12.56 mL H₂.h^?1, respectively. The temperature 37 °C and pH 7.0 were the optimum conditions for P (70.19 mL H₂) and Rm (12.43 mL H₂.h^?1) as predicted by the mathematical model. Fructose and glucose are the primary alternative carbon sources in banana waste-fed batch reactors. The high concentration of lactic acid and H₂ production was associated to Lactobacillus (52–81%) and Clostridium (14–35%). However, the most important finding was about butyric acid (HBu). This acid is the better indicator of hydrogen production than acetic acid (HAc). The pH effected carbohydrates fermentation and organic acids production. The genes encoding the enzymes related to galactose, sucrose, fructose, arabinose and xylose metabolism were predominant.     

Fermentative hydrogen production by diverse microflora

In this study, hydrogen production with activated sludge, a diverse bacterial source has been investigated and compared to microflora from anaerobic digester sludge, which is less diverse. Batch experiments were conducted at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The hydrogen production yields with activated sludge at 37 °C and 55 °C were 0.56 and 1.32 mol H₂/mol glucose consumed, respectively. While with anaerobically digested sludge hydrogen yield was 2.18 mol H₂/mol glucose consumed at 37 °C and 1.25 mol H₂/mol glucose consumed at 55 °C. The results of repeated batch experiments for 615 h resulted in average yields of 1.21 ± 0.62 and 1.40 ± 0.16 mol H₂/mol glucose consumed for activated sludge and anaerobic sludge, respectively. The hydrogen production with activated sludge was not stable during the repeated batches and the fluctuation in hydrogen production was attributed to formation of lactic acid as the predominant metabolite in some batches. The presence of lactic acid bacteria in microflora was confirmed by PCR-DGGE.     

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.     

The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures

The effect of heat treatment at different temperatures on two types of inocula, activated sludge and anaerobically digested sludge, was investigated in batch cultures. Heat treatments were conducted at 65, 80 and 95 °C for 30 min. The untreated inocula produced less amount of hydrogen than the pretreated inocula, with lactic acid as the main metabolite. The maximum yields of 2.3 and 1.6 mol H₂/mol glucose were achieved for the 65 °C pretreated anaerobically digested and activated sludges, respectively. Approximately a 15% decrease in yield was observed with increasing pretreatment temperature from 65 to 95 °C concomitant with an increase in butyrate/acetate ratio from 1.5 to 2.4 for anaerobically digested sludge. The increase of pretreatment temperature of activated sludge to 95 °C suppressed the hydrogen production by lactic acid fermentation. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity.     

The feasibility of using geothermal energy in hydrogen production

A feasibility study exploring the use of geothermal energy in hydrogen production is presented. It is possible to use a thermal energy to supply heat for high temperature electrolysis and thereby substitute a part of the relatively expensive electricity needed. A newly developed HOT ELLY high temperature steam electrolysis process operates at 800 – 1000°C. Geothermal fluid is used to heat fresh water up to 200°C steam. The steam is further heated to 900°C by utilising heat produced within the electrolyser. The electrical power of this process is reduced from 4.6 kWh per normalised cubic meter of hydrogen (kWh/Nm³ H₂) for conventional process to 3.2 kWh/Nm³ H₂ for the HOT ELLY process implying electrical energy reduction of 29.5%. The geothermal energy needed in the process is 0.5 kWh/Nm³ H₂. Price of geothermal energy is approximately 8–10% of electrical energy and therefore a substantial reduction of production cost of hydrogen can be achieved this way. It will be shown that using HOT ELLY process with geothermal steam at 200°C reduces the production cost by approximately 19%.     

Continuous fermentative hydrogen production from coffee drink manufacturing wastewater by applying UASB reactor

The feasibility of continuous H₂ production from coffee drink manufacturing wastewater (CDMW) was tested in two different types of reactors: a completely-stirred tank reactor (CSTR) and an up-flow anaerobic sludge blanket reactor (UASBr). While the performance in CSTR was limited, it was significantly enhanced in UASBr. The maximum H₂ yield of 1.29 mol H₂/mol hexose_added was achieved at HRT of 6 h in UASBr operation. Non-hydrogenic, lactic acid was the dominant in CSTR, while butyric and caproic acids in UASBr. As caproic acid is generated by consuming acetic and butyric acids, all of which are related to H₂ production, the presence of caproic acid in the broth also indicates H₂ production, yielding 1.33 mol H₂/glucose. It was speculated that the enhanced performance in UASBr was attributed to the high concentration of biomass over 60,000 mg VSS/L in the blanket zone, which provided insufficient substrate for indigenous lactic acid bacteria (LAB) to survive. The abundance of LAB in CDMW was confirmed by natural fermentation of CDMW. That is without the addition of external inoculum, CDMW was mainly fermented into lactic acid under mesophilic condition. For the first time ever, H₂ producing granules (HPG) with diameters of 2.1 mm were successfully formed by using actual waste as a substrate.     

Hydrogen,alcohols and volatile fatty acids from the co-digestion of coffee waste (coffee pulp,husk, and processing wastewater) by applying autochthonous microorganisms

The objective of this study was to screen the factors that affect H₂, organic acids and alcohols production from coffee waste pretreated in a hydrothermal reactor applying consortium of bacteria and fungi (indigenous from coffee waste) with hydrolytic and fermentative activity. The effects of pH (4.0–7.0), temperature (30–50 °C), agitation (0–180 rpm), headspace (50–70%), percentage of bioaugmentation (without microbial consortium to 20%), concentration of coffee pulp and husk (2–6 g/L), coffee processing wastewater (7-30 gCOD/L) and yeast extract (0–2 g/L) were evaluated using a Plackett-Burman design. The highest H₂ production potential (82 ml H₂) was obtained under the following conditions: 30 °C, 180 rpm, 50% headspace, without bioaugmentation, 2 g/L pulp and husk coffee, 30 gCOD/L coffee processing wastewater and 2 g/L yeast extract. The main soluble products were acetic acid (1956 mg/L), lactic acid (786 mg/L) and ethanol (816 mg/L). Lactobacillus sp., Clostridium sp., Saccharomyces sp. and Kazachstania sp. were the main autochthonous microorganisms identified. Through metagenome functional analysis, enzymes related to lignin, phenol, cellulose, lignocellulose, and pectin degradation were identified, as well as acidogenesis, and H₂ production.     

Microbial ecology of a lactate-driven dark fermentation process producing hydrogen under carbohydrate-limiting conditions

Despite the high prevalence of lactic acid bacteria in dark fermentation (DF) processes, their ecological role is not yet completely elucidated, preventing their systematic use as “helpers” for hydrogen production. The aim of this study was to investigate the microbial community structure of a lactate-driven DF process that successfully produced hydrogen under carbohydrate-limiting conditions using tequila vinasse as a substrate. Microbial responses to stepwise decreases in hydraulic retention time (HRT) from 24 to 4 h were assessed by using Illumina MiSeq sequencing. HRTs above 12 h and below 6 h led to a lower hydrogen production rate (HPR; 0.2–3.3 L/L-d) and process stability (HPR variations within 25–65%), which were associated with the presence of Acetobacter lovaniensis, Clostridium luticellari, Blautia coccoides, and the high abundance of propionate and lactate. Interestingly, transient conditions from unsteady-to-steady state occurred at an HRT of 12 h, where species richness and evenness decreased remarkably. Accordingly, HRTs between 12 and 6 h resulted in higher HPRs of up to 11.7 ± 0.7 L/L-d with HPR variations of less than 10%, which closely matched with the dominance of Clostridium sp., and butyrate and acetate as the main aqueous products. Overall, the results indicate that the successfulness of exploiting the ‘unwanted’ LAB proliferation through lactate-driven DF processes requires the enrichment of lactate-consuming and hydrogen-producing bacteria, which entails the selection of proper biocatalysts and operating conditions/strategies such as the operation of DF reactors under carbohydrate-limiting conditions and low HRTs.     

Optimization of biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent using response surface methodology

Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P_c) and maximum hydrogen production rate (R_max) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P_c) were pH 5.69, 36 °C, and 92 g COD/l; with an estimated P_c value of 306 ml H₂/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R_max) were pH 6.52, 41 °C and 60 g COD/l; with an estimated R_max value of 914 ml H₂/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 °C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%.