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Evaluation of methods for preparing hydrogen-producing seed inocula under thermophilic condition by process performance and microbial community analysis
Abstract
Five methods for preparation of hydrogen-producing seeds (base, acid, 2-bromoethanesulfonic acid (BESA), load-shock and heat shock treatments) as well as an untreated anaerobic digested sludge were compared for their hydrogen production performance and responsible microbial community structures under thermophilic condition (60 degrees C). The results showed that the load-shock treatment method was the best for enriching thermophilic hydrogen-producing seeds from mixed anaerobic cultures as it completely repressed methanogenic activity and gave the a maximum hydrogen production yield of 1.96 mol H(2) mol(-1) hexose with an hydrogen production rate of 11.2 mmol H(2) l(-1)h(-1). Load-shock and heat-shock treatments resulted in a dominance of Thermoanaerobacterium thermosaccharolyticum with acetic acid and butyric acid type of fermentation while base- and acid-treated seeds were dominated by Clostridium sp. and BESA-treated seeds were dominated by Bacillus sp. The comparative experimental results from hydrogen production performance and microbial community analysis showed that the load-shock treatment method was better than the other four methods for enriching thermophilic hydrogen-producing seeds from anaerobic digested sludge. Load-shock treated sludge was implemented in palm oil mill effluent (POME) fermentation and was found to give maximum hydrogen production rates of 13.34 mmol H(2) l(-1)h(-1) and resulted in a dominance of Thermoanaerobacterium spp. Load-shock treatment is an easy and practical method for enriching thermophilic hydrogen-producing bacteria from anaerobic digested sludge.
... It is feasible to use an anaerobic mixed culture as inoculum for acidogenic fermentation, but methanogenic activity must be suppressed [13]. For this goal, there are several conditioning methods reported in the literature: chemical inhibitors such as 2-bromoethanesulfonate acid, methyl chloride and lumazine [6,14]; chemical conditioning such as acid, alkali and organic shock addition; and physical strategies such as heat, freezing and thawing, ultrasound, among others that effectively inhibit methanogens [13,15]. ...
... To achieve favourable conditions for acidogenic fermentation, methanogenic activity should be inhibited [31]. Inoculum conditioning methods, such as heat-shock, acid and alkali treatments, have been shown to effectively repress methanogenic activity [14]. Therefore, these three methods were evaluated to identify the optimal treatment for our purposes. ...
... The highest degree of acidification was obtained for heat shock (39 ± 2%), followed by the alkali pre-conditioning method (35 ± 2%), while the lowest degree of acidification (22 ± 1%) was obtained for acid pre-conditioning. In this sense, O-Thong et al. [14] and Zhang et al. [26] reported that heat-shock conditioning was the most preferable method for suppressing methanogenic activity, while the results for the acid and alkali conditioning methods were contradictory between the two works. However, Ren et al. [32] reported the following order for the degree of acidification: alkali > heat-shock > acid conditioning, which is in agreement with our results. ...
Acidogenic fermentation of wastes produces volatile fatty acid (VFA)-rich streams that can be used as low-cost carbon sources for polyhydroxyalkanoate (PHA) production. In this study, an inoculum collected from an anaerobic reactor of a municipal WWTP was conditioned to suppress methanogenic activity. The heat-shock conditioning method of the inoculum proved to be more efficient than acid and alkaline conditioning methods for methanogens inhibition. Then, the pre-conditioned inoculum was used to determine the acidogenic potential of different wastes: three waste activated sludge (WAS) samples generated at different sludge retention times (SRTs, 2, 7 and 14 days), olive mill wastewater (OMW), glycerol, apple pomace (AP) and winterization oil cake (WOC). Batch tests were performed in quintuplicate at 37 °C and pH 7. A higher degree of acidification was observed for high-rate activated sludge (2 days of SRT) (69%), followed by olive mill wastewater (OMW) (43%), while the lowest was for glycerol (16%). The results for the winterization oil cake (WOC) samples interestingly elucidated a high content of propionic acid with a high odd-to-even ratio (0.86) after fermentation. Feeding the VFA profile obtained from WOC into a PHA production system led to a significant production of 0.64 g PHA g⁻¹ C with 30% polyhydrobutyrate (PHB) to 69% polyhydroxyvalerate (PHV) as monomeric units of HB-co-HV, decoupling the need for a related carbon source for co-polymer production.
Graphic Abstract
... It is feasible to use an anaerobic mixed culture as inoculum for acidogenic fermentation, but methanogen activity must be suppressed [13]. For this goal, there are several conditioning methods reported in the literature: chemical inhibitors such as 2-bromoethanesulfonate acid, methyl chloride and lumazine [6,14]; chemical conditioning such as acid, alkali and organic shock addition; and physical strategies such as heat, freezing and thawing, ultrasound, among others that effectively inhibit methanogens [13,15]. ...
... 3.1 Comparison of the effect of the inoculum conditioning methods on VFA bioconversion and H 2 production To achieve favourable conditions for acidogenic fermentation, methanogenic activity should be inhibited [31]. Inoculum conditioning methods, such as heat-shock, acid and alkali treatments, have been shown to effectively repress methanogenic activity [14]. Therefore, these three methods were evaluated to identify the optimal treatment for our purposes. ...
... The highest degree of acidi cation was obtained for heat shock (39 ± 2%), followed by the alkali pre-conditioning method (35 ± 2%), while the lowest degree of acidi cation (22 ± 1%) was obtained for acid pre-conditioning. In this sense, O-Thong et al. [14] and Zhang et al. [26] reported that heat-shock conditioning was the most preferable method for suppressing methanogenic activity, while the results for the acid and alkali conditioning methods were contradictory between the two works. However, Ren et al. [32] reported the following order for the degree of acidi cation: alkali> heat-shock> acid conditioning, which is in agreement with our results. ...
Acidogenic fermentation of wastes produces volatile fatty acid (VFA)-rich streams that can be used as low-cost carbon sources for polyhydroxyalkanoate (PHA) production. In this study, an inoculum collected from an anaerobic reactor of a municipal WWTP was conditioned to suppress methanogenic activity. The heat-shock conditioning method of the inoculum proved to be more efficient than acid and alkaline conditioning methods for methanogen inhibition. Then, the pre-conditioned inoculum was used to determine the acidogenic potential of different wastes: three waste activated sludge (WAS) samples generated at different sludge retention times (SRTs, 2, 7 and 14 days), olive mill wastewater (OMW), glycerol, apple pomace (AP) and winterization oil cake (WOC). Batch tests were performed in quintuplicate at 37°C and pH 7. A higher degree of acidification was observed for high-rate activated sludge (2 days of SRT) (69%), followed by olive mill wastewater (OMW) (43%), while the lowest was for glycerol (16%). The results for the winterization oil cake (WOC) samples interestingly elucidated a high content of propionic acid with a high odd-to-even ratio (0.86) after fermentation. Feeding the VFA profile obtained from WOC into a PHA production system led to a significant production of 0.64 g PHA g − 1 C with 30% polyhydrobutyrate (PHB) to 69% polyhydroxyvalerate (PHV) as monomeric units of HB-co-HV, decoupling the need for a related carbon source for co-polymer production.
... Hydrogen-producing sludge for the first-stage was collected from a hydrogen production reactor feeding with palm oil mill effluent. The sludge was cultivated on a 2 g L −1 sucrose medium for enhancing hydrogen-producing bacteria (O-Thong, Prasertsan & Birkeland, 2009). The enriched sludge with volatile suspended solids of 6.0 g L −1 was used as inoculum for the first stage (Mamimin et al., 2015). ...
... PCR amplification was conducted in an automated thermal cycler with pre-denaturation at 95 • C for 5 min followed by 25 cycles of denaturation at 95 • C for 30 s, annealing at 52 • C for 40 s, elongation at 72 • C for 90 s, and post-elongation at 72 • C for 5 min. The reactions were subsequently cooled to 4 • C (O- Thong, Prasertsan & Birkeland, 2009). The second PCR was amplified from the amplicons of the first PCR as a DNA template with primer Arch519r (5 TTACCGCGGCKGCTG 3 with 40 bp GC clamp) and Arch340f (5 CCTACGGGGYGCASCAG 3 ) for archaea population. ...
... PCR products were analyzed on agarose gel electrophoresis before DGGE analysis. The amplicons from the second PCR were used for DGGE analysis, as previously described by Prasertsan, O-Thong & Birkeland (2009). The DGGE bands were excised from the gel and re-amplified under similar conditions as the second PCR. ...
Background
Anaerobic digestion (AD) is a suitable process for treating high moisture MSW with biogas and biofertilizer production. However, the low stability of AD performance and low methane production results from high moisture MSW due to the fast acidify of carbohydrate fermentation. The effects of organic loading and incineration fly ash addition as a pH adjustment on methane production from high moisture MSW in the single-stage AD and two-stage AD processes were investigated.
Results
Suitable initial organic loading of the single-stage AD process was 17 gVS L ⁻¹ at incineration fly ash (IFA) addition of 0.5% with methane yield of 287 mL CH 4 g ⁻¹ VS. Suitable initial organic loading of the two-stage AD process was 43 gVS L ⁻¹ at IFA addition of 1% with hydrogen and methane yield of 47.4 ml H 2 g ⁻¹ VS and 363 mL CH 4 g ⁻¹ VS, respectively. The highest hydrogen and methane production of 8.7 m ³ H 2 ton ⁻¹ of high moisture MSW and 66.6 m ³ CH 4 ton ⁻¹ of high moisture MSW was achieved at organic loading of 43 gVS L ⁻¹ at IFA addition of 1% by two-stage AD process. Biogas production by the two-stage AD process enabled 18.5% higher energy recovery than single-stage AD. The 1% addition of IFA into high moisture MSW was useful for controlling pH of the two-stage AD process with enhanced biogas production between 87–92% when compared to without IFA addition. Electricity production and energy recovery from MSW using the coupled incineration with biogas production by two-stage AD process were 9,874 MJ ton ⁻¹ MSW and 89%, respectively.
Conclusions
The two-stage AD process with IFA addition for pH adjustment could improve biogas production from high moisture MSW, as well as reduce lag phase and enhance biodegradability efficiency. The coupled incineration process with biogas production using the two-stage AD process was suitable for the management of MSW with low area requirement, low greenhouse gas emissions, and high energy recovery.
... Heat, acid, and alkali pretreatments were applied to the inoculum to limit methanogenic activity. For acid and alkali pretreatment, the sludge was adjusted to pH 3.0 or 12.0 using 1 M HCl acid or NaOH, respectively [9,14]. It was then mixed and stirred at 37 • C and 200 rpm for 24 h, followed by readjusting the pH back to 7.0. ...
... Montiel-Jarillo et al. [9] reported the following order for the hydrolysis efficiency: heat > alkali > acid, which is in agreement with our results. Heat pretreatment has been regarded as an effective inoculum pretreatment strategy to selectively enrich sporulating acidogenic bacteria and suppressing methanogenic activity [14,23]. ...
Inoculum pretreatment and substrate/inoculum ratio (SIR) are essential factors affecting the acidogenic fermentation of chemically enhanced primary treatment (CEPT) sludge. To determine the optimal inoculum conditions, the influence of inoculum pretreatment and SIR on the production of volatile fatty acids (VFAs) was investigated via two phases of batch experiments. Heat, acid, and alkali pretreatment methods demonstrated the enhanced production of VFAs, with the heat pretreatment being the optimal inoculum pretreatment method due to its highest VFA accumulation and favorable VFA composition for denitrification. The substrate/inoculum ratio of 4:1 (SIR 4) presented the optimal efficiency for both hydrolysis and acidogenesis processes (24.6 ± 0.1% and 22.7 ± 0.4%), with acetic acid, butyric acid, and propionic acid dominating the VFA profile. Combining VFA production and microbial community, the heat-pretreated inoculum with the SIR 4 condition was the most suitable for the VFA production of CEPT sludge acidogenic fermentation. This study contributes to sustainability in wastewater management by demonstrating an efficient approach for the recovery of carbon resources from CEPT sludge. The optimized conditions for acidogenic fermentation not only enhance VFA production but also support the circular economy by transforming waste into valuable resources.
... Therefore, an effective pretreatment is required to inhibit the H 2 consuming bacterial activity, as well as enrich anaerobic spore-forming bacteria. Usually, pretreatment methods include heat (O-Thong et al. 2009), acid and base stock and base shock (O-Thong et al. 2009;Yang and Wang 2018), and electric field (Jeong et al. 2013). However, heat shock microbial culture has the best performance in a higher yield of H 2 production. ...
... Therefore, an effective pretreatment is required to inhibit the H 2 consuming bacterial activity, as well as enrich anaerobic spore-forming bacteria. Usually, pretreatment methods include heat (O-Thong et al. 2009), acid and base stock and base shock (O-Thong et al. 2009;Yang and Wang 2018), and electric field (Jeong et al. 2013). However, heat shock microbial culture has the best performance in a higher yield of H 2 production. ...
Biohydrogen is considered a fuel for the future due to its unique attributes in clean energy generation, waste management, and high energy content. Recently, its economic production has gained considerable attention from numerous scientists and industrialists. This chapter addresses microbiological, biochemical, molecular biological, and other perspectives related to biological hydrogen production (BHP). Process parameters such as pH, substrate type, temperature, agitation speed, hydraulic retention time, and hydrogen partial pressure greatly influence the dark fermentation process. Therefore, several optimization approaches, including statistical and artificial intelligence, have been demonstrated. Additionally, different kinetic models associated with substrate degradation, cell mass growth, and product formation in dark fermentation have been discussed in detail. This chapter also discusses different types of reactors and their suitability for biological hydrogen production. The viability of any process relies on its ability to be applied to the industrial level. Therefore, the scale-up of the biohydrogen production process has been exemplified. In summary, this chapter presents a holistic overview of the biohydrogen production process and highlights recent scientific findings and achievements.
... Posee el máximo rendimiento energético por unidad de peso, siendo fácil almacenar y de transportar. Además, el H 2 es limpio, produce sólo agua cuando se quema, eliminando los problemas de contaminación atmosférica y efecto invernadero que tienen los combustibles fósiles Liu y Shen, 2004;Thong et al, 2008;Jo et al, 2008;Pan et al, 2008;Wang y Wan, 2008;Oztekin et al, 2008). El H 2 se genera en la actualidad mediante diferentes procesos: reformado de gas natural, gasificación de carbón y pirólisis, que utilizan combustibles fósiles no renovables y requieren un aporte de energía, o procesos electroquímicos, que tienen un elevado consumo energético. ...
... In most studies, anaerobic digestion sludge, aerobic compost, sewage water treatment sludge, agricultural soil, river sediment, sludge compost and isolated bacteria have been used as inocula for hydrogen fermentation (Kawagoshi et al, 2005;Koskinen et al. 2008;Vazquez et al, 2008;Ohnishi et al, 2010) (Figure 2.7). Previous works have reported different methods for preparation of hydrogen-producing seeds (base, acid, 2-bromoethanesulfonic acid, load shock and heat shock treatments) (Thong et al, 2008). In most cases, inocula are conditioned by heating or pH treatment to enhance hydrogen production because hydrogen producing bacteria are commonly tolerant to extreme environmental conditions and methanogens can be rid (Tang et al., 2008). ...
1) Ten hydrogen-producing strains affiliated to the Clostridium genus were isolated from different sources.
2) An enrichment culture [EC] was obtained from granular sludge after growth during five months under vacuum conditions. The bacterial community of the EC included Enterococcus, Delftia, Bacillus and Bacteroides.
3) Optimal growth and hydrogen production by dark fermentation for the isolated strains and EC was achieved in most cases at an initial pH of 6.5 and at a temperature of 35 °C.
4) The hydrogen production of the different strains depended on the substrate used along with their saccharolytic and proteolytic properties. C. saccharobutylicum H1 and C. butyricum R4 displayed the best hydrogen production using glucose as the sole carbon source. C. roseum H5 and C. butyricum R6 had the best yields growing on meat extract. For complex media, C. roseum H5 and C. diolis RT2 had the highest hydrogen production, with values of 120 mL-H2 g-1 initial COD. When COD removed is considered, C. beijerinckii UAM and C. diolis RT2, reached 573 and 475 mL-H2 g-1 removed COD.
5) The different strains studied carried out the fermentation of substrates through different metabolic pathways, with butyric acid fermentation being widespread. Butyrate and acetate were the main end products followed by propionate, lactate and ethanol. In addition, the end-product ratios were strongly dependent on the strain, pH, temperature and substrate.
6) C. saccharobutylicum H1 degraded glucose by butyric fermentation following a pseudo-first order kinetic. The kinetical parameters for biomass growth and glucose consumption were accurately described and the generation time estimated to be 1.87 h. When the pH dropped below 4, acetoacetate was excreted into the media.
7) Anaerobic granular sludge carried out a wide variety of fermentations: alcoholic, lactic (hetero and homo), butyric, propionic, and mixed acid depending on the culture media used. An obvious metabolic shift took place in response to the application of vacuum. Moreover, the vacuum improved the mineralization of organic matter.
8) DGGE profiles suggest a wide array of microbial populations, with shifts in the dominant taxa depending on the substrate available and the presence of vacuum. The genera Clostridium, Klebsiella, Acetobacter, Arcobacter, Desulfovibrio, Dysgonomonas and Azospira were observed in the Bacteria domain, and Methanosaeta and Methanosarcina were found in the Archaea domain.
9) Hydrogen production can be increased with the use of co-cultures. C. butyricum R4 and C. roseum H5 proved to be the best combination for hydrogen production of the co-cultures studied.
10) The development of a consortium between hydrogen-producing clostrdia and Streptomyces sp. could be useful for future industrial applications because Streptomyces (i) removes the oxygen present in the influent, avoiding the use of reducing agents and enhancing hydrogen production, and (ii) forms granules that embed the hydrogen-producers, favoring the accumulation of large amounts of biomass inside a bioreactor.
11) The inclusion of Syntrophobacter wolinii and Methanosaeta concilii in the former consortium could remove the butyrate and acetate accumulated obtaining, in a second step after the hydrogen recovery, methane.
... None of the physical methods mentioned above is universal, ensuring a high hydrogen production yield. Inocula originating from dierent sources require different pretreatment methods, which was the subject of numerous investigations [204][205][206][207][208][209][210]. Pretreatment methods also inhibit strains of bacteria not forming spores and producing hydrogen, such as Enterobacter, resulting in a lower hydrogen yield compared with inoculum that had not been pretreated [201]. ...
... The advantage of granulated form of microorganisms is their higher retention in the reactor and higher resistance to toxic conditions. [26,27,134,202,208]. [42,194,[209][210][211]. ...
Hydrogen applicability in the power, chemical and petrochemical industries is constantly growing. Efficient methods of hydrogen generation from renewable sources, including waste products, are currently being developed, even though hydrogen is mainly produced through steam reforming or thermal cracking of natural gas or petroleum fractions. In paper alternative methods of hydrogen production with a particular emphasis on dark fermentation are discussed. The review compiles essential information on strains of bacteria used in the production of hydrogen from waste products in the agroindustry and from lignocellulosic biomass. The effect of such parameters as kind of raw material, method of processing, temperature, pH, substrate concentration, partial pressure of hydrogen, hydraulic retention time, method of inoculum preparation and the type and operating parameters of a reactor on the yield of dark fermentation is discussed. The review aims at presentation of current state of knowledge on the dark fermentation process utilizing waste materials as substrates. The results of investigations with emphasis on the most important issues regarding operating parameters of dark fermentation are also included.
... In the biohydrogen production, it was determined that the methanogenic activity is fully inhibited when applying heat shock at 105°C for 1 h with glucose as a substrate [13], as well as at 70°C for 1 h assisted with free ammonia at 131.9 and 168.3 mg L −1 using waste activated sludge as inoculum and substrate [14]. On the other hand, heat shock pretreatment increased the hydrogen production ability of anaerobic sludge in dark fermentation up to 6.2 (65°C for 30 min), 4.2 (100°C for 1 h), and 6.8 (100°C for 1 h)-fold more with substrates such as glucose [6], sucrose [15], and dairy water [8], respectively. Ultrasound (US) has extended its use in several technological applications and research areas due to physical effects such as cavitation and mechanisms linked to it, such as microstreaming and sonoporation. ...
... The highest methane production inhibition on the anaerobic sludge was reached in the HS pretreatment (96.4 ± 0.6%), followed by the US pretreatment in the USE intervals of 41 to 102.5 kJ L −1 (82.3 ± 0.29 to 88.9 ± 0.17%), and finally the USE of 20.5 kJ L −1 (77.9 ± 0.37%). Similar inhibition levels on the anaerobic sludge with regard to producing methane have been observed by ultrasound application for 5 min at 20 kHz and with temperature control [24], as well as with heat shock at 100°C for 1 h [15], 121°C for 1 h [8], and 70°C for 30 min [24]. On the other hand, the anaerobic sludge displayed greater hydrogen production than the control when pretreated with US to several USE levels as well as with HS (Fig. 1b). ...
In the present work, the ultrasonic specific energy (USE) effect on anaerobic sludge in methane inhibition and hydrogen production capability by dark fermentation in pretreatments with ultrasound (US) and combined ultrasound with heat shock (US + HS) was evaluated. The non-pretreated anaerobic sludge was used as a control, and the sludge pretreated with heat shock (HS) at 85 °C for 45 min was a comparison system. HS inhibited methane production by 96.4 ± 0.6%, while pretreatments with US and US + HS in a USE interval between 20.5 and 102.5 kJ L−1 showed inhibition of 77.9 ± 0.37 to 88.9 ± 0.17% and 89.8 ± 0.17 to 95.7 ± 0.10%, respectively. Nevertheless, from the statistical analysis, it was determined that the HS and US + HS in all of the USE levels have the same inhibition effect. US at 41.0 kJ L−1 reached an accumulated hydrogen value that was 3.2-fold higher than the control and 79.5% greater than HS, while for US + HS at 82.0 kJ L−1, it was 2.3-fold higher than the control and 19.6% greater than HS. A comparative analysis from adimensionalized kinetic parameters showed that hydrogen production potential (Pmax) and maximum hydrogen rate (Rmax) on the anaerobic sludge pretreated with US and US + HS at 40 kHz are higher than or similar to that reported. The higher efficiencies of anaerobic sludge in hydrogen production from sucrose were seen in USE levels of 41.0 and 61.5 kJ L−1 for US, and of 61.5 and 82.0 kJ L−1 for US + HS. The HAc/HBut molar ratio from 0.255 to 0.410 showed that hydrogen was produced via the butyrate route. The ultrasonic specific energy applied to the anaerobic sludge of 41.0–61.5 kJ L−1 by ultrasound and 61.5–82.0 kJ L−1 by combined ultrasound with heat shock is a highly feasible method with which to inhibit methane production and improve hydrogen production in dark fermentation.
... For example, heat, acid, and base pretreatment were favorable to enriching the hydrogen producers, like Clostridium spp., Enterococcus spp., and Bacillus spp. (Liu et al. 2009); Bacillus species predominated in the mixed culture that was treated with 2-bromo-ethano-sulfonic acid (BESA) (O-Thong et al. 2009). In addition to the techniques of treatment, the sources of the inoculum play a significant role on the diversity of microbes found in a fermentation system. ...
Direct or indirect biophotolysis, photo-fermentation, and dark fermentation are all methods of biohydrogen production (BHP), with the latter being the only one that doesn’t require the addition of light energy. The significant research on the application of pure and defined coculture dark fermentative biohydrogen production is compiled in this chapter. The recent advances in microbiology, including biochemistry, enzymology, microbial modification, and the identification of the microbial community structure, for the production of hydrogen through dark fermentation are discussed. By altering metabolic pathways, metabolic engineering can improve the biological production of hydrogen by removing constraints on hydrogen synthesis in various systems, increasing electron flow to hydrogen-producing pathways, boosting substrate utilization, and designing more effective enzymes. Moreover, leading biohydrogen-producing microbes like Clostridium spp., Escherichia coli, Enterobacter spp., Bacillus spp., etc. are also covered, as well as innovative methods used to increase biohydrogen production.
... Clostridium and Thermoanaerobacterium are the most common bacteria employed during dark fermentation for the production of biohydrogen. Furthermore, multiple investigations have shown that mixed cultures in batch or in bioreactors can produce biohydrogen (Shin et al. 2004;O-Thong et al. 2009;Ismail et al. 2010;Prasertsan et al. 2009;Ghimire et al. 2015). The benefits of utilising combined cultures for biohydrogen production includes no sterilisation, high adaptive ability of microbial diversity, the ability to make use of a mixture of substrates, and the potential of acquiring a steady and continuous course of biohydrogen production (Ismail et al. 2010). ...
The microbial fermentation process or MFP is a technique used in several sectors to produce natural, novel, eco-friendly, and pragmatical products for human beings. The MFP technique has been extensively studied and applied in pharmaceutical, dairy, fruit juice, and agricultural sectors and industries. Consequently, by-products in the form of solid and liquid wastes are generated in various sectors and business establishments, making waste management difficult. Hence, for the management of the waste generated by these industries, the by-products were used as a substrate for producing biopolymers and bioenergy by the action of microbes. Moreover, microbes utilise these by-products generated by various industries in their metabolic pathway to produce biopolymers and bioenergy as end products during fermentation processes. The aerobic fermentation process has been mainly used for biopolymer production, and the anaerobic fermentation process is used for bioenergy, such as biogas and bio-hydrogen. Several microbes have been reported, such as Bacillus spp., Nocardia spp., methylotrophs, Alcaligenes spp., Rhizobium spp., Azotobactor spp., Pseudomonas spp., and recombinant Escherichia coli, by researchers in their research work. This chapter summarises the conversion of the complex substrate (waste) to the transparent substrate (waste), microbial strains, and fermentation techniques to produce biopolymers and bioenergy. This information is beneficial for selecting a suitable substrate source for a particular product generation with a known fermentation process and/or modifying the existing fermentation process.
... [60,61], Thermoanaerobacterium sp. [62], Ethanoligenens sp. [63], etc.) facultative anaerobes (e.g., Bacillus sp. ...
Saccharina japonica (known as Laminaria japonica or Phaeophyta japonica), one of the largest macroalgae, has been recognized as food and medicine for a long time in some Asian countries, such as China, South Korea, Japan, etc. In recent years, S. japonica has also been considered the most promising third-generation biofuel feedstock to replace fossil fuels, contributing to solving the challenges people face regarding energy and the environment. In particular, S. japonica-derived biohydrogen (H2) is expected to be a major fuel source in the future because of its clean, high-yield, and sustainable properties. Therefore, this review focuses on recent advances in bio-H2 production from S. japonica. The cutting-edge biological technologies with suitable operating parameters to enhance S. japonica’s bio-H2 production efficiency are reviewed based on the Scopus database. In addition, guidelines for future developments in this field are discussed.
... Pretreatment of substrate involving physical treatment such as load shock treatment of heat can contribute to the dominance of hydrogen producer over hydrogen consumers to increase biohydrogen production yield [48]. Besides, methanogenic inhibitors such as 2-bromoethanesulfonate and iodopropane can be employed as a chemical pretreatment against methanogens [49]. ...
Depletion of fossil fuels and alarming global warming phenomena due to greenhouse gases emission into the atmosphere have led to the high demand for alternative and clean energy sources. The production and utilization of H 2 as an eco‐friendly energy source is crucial for combatting earlier issues. However, despite being a very clean energy source, the current hydrogen generation system, such as steam reforming, is considered not an environmental friendly approach. Thus, anaerobic digestion has been a promising method to produce hydrogen from livestock, crops, and food waste. Therefore, this chapter recapitulated the fundamentals behind anaerobic digestion to produce hydrogen and highlighted the challenges and mitigation strategies in biohydrogen production. Finally, the practicality of anaerobic digestion technologies at an industrial scale is discussed.
... In this method, the mixed cultures are exposed to a high concentration of carbon source in which a diversity of hydrogen producers are enriched. This method is favored to enrich the hydrogen producer and eliminate the methane-producing archaea [22,23]. The inoculum for methane production (INM) was collected from the same location, but it was not pretreated before use as the inoculum. ...
This study aimed to enhance dark fermentative hydrogen production from co-digestion of distillery wastewater (DW) and glycerol waste (GW) through integration with microbial electrolysis cells. First, the optimal proportion of DW and GW in hydrogen production was investigated in batch mode. The results show that DW and GW co-digestion at a ratio of 99:1 (% v/v) gave the highest hydrogen yield of 149.5 mL-H2/g-VSadded. Continuous hydrogen production using the optimal proportion was conducted in a continuously stirred tank reactor. As a result, a maximal hydrogen yield of 99.7 mL-H2/g-VSadded was achieved, and the dominant hydrogen-producing bacterium was Clostridium sensu stricto 7. The dark fermentation effluent from the continuously stirred tank reactor was later used to produce methane using batch MECs. The maximum methane yield of 115.1 mL-CH4/g-VSadded was obtained under an applied voltage of 1 V and continuous stirring at 120–140 rpm. Microbial community analysis revealed that Metahnobacterium, Methanomethylovorans, Methanoculleus, and Methanosarcina were the methanogenic archaea in the microbial electrolysis cell reactor.
... Acid pretreatment was also commonly applied to suppress methanogens. 136 For example, Wang et al. 137 and O-Thong et al. 138 reported that the H 2 production rate of acidpretreated sludge increased by 72% and 212% compared with that of the untreated group, respectively. ...
Hydrogen gas (H2) is an attractive fuel carrier due to its high specific enthalpy; moreover, it is a clean source of energy because through combustion reaction with oxygen (O2), it produces water as the only byproduct. Microbial electrolysis cell (MEC) is a promising technology for producing H2 from simple or complex organics present in wastewater and solid wastes. Methanogens and non-archaeal methane (CH4)-producing microorganisms (NAMPMs) often grow in the MECs, which lead to rapid conversion of produced H2 to CH4 in the MECs. Moreover, non-archaeal methane production (NAMP) catalyzed by nitrogenase of photosynthetic bacteria was always overlooked. Thus, suppression of CH4 production is required to enhance H2 yield and production rate. This review comprehensively addresses the principles and current state-of-the-art technologies for suppressing methanogenesis and NAMP in MECs. Noteworthy, specific strategies aiming at the inhibition of methanogenic enzymes and nitrogenase could be a more direct approach than physical and chemical strategies for repressing the growth of methanogenic archaea. In-depth studies on the multi-omics of CH4 metabolism can possibly provide insights into sustainable and efficient approaches for suppressing metabolic pathways of methanogenesis and NAMP. The main objective of this review is to highlight key concepts, directions, and challenges related to boosting H2 generation by suppressing CH4 production in MECs. Finally, the perspectives are briefly outlined to guide and advance the future directions of MECs for production of high-purity H2 based on genetic and metabolic engineering, and on the interspecific interactions.
... To enrich the hydrogen producers in the sludge, methanogens need to be eliminated from the inoculum and that is where the aid of seed pretreatment is required. There are various types of seed pretreatment methods: chemical treatment, heat-shock treatment, and organic load-shock treatment (O-Thong et al. 2009). Chemical treatments involve either acid or alkali to eliminate methanogens at extreme pH. ...
Antibiotics are bioactive compounds that selectively kill or mitigate the growth of microorganisms. The increasing human population, development, and industrialization resulted in an increased demand of antibiotics. The annual consumption of antibiotics round the globe has reached over 200,000 tons. Hence, alternative and cost-effective sources for the production of antibiotics are inevitable. Agricultural wastes, i.e., corn cobs, sawdust, rice hulls, and groundnut shell, are rich source of bioactive compounds. Therefore, the agro-waste can be utilized for industrial production of various value-added products including antibiotics. The composition, quantity, and quality of antibiotics produced from agro-waste depend on both starting material/substrate (raw waste) and the processing steps. By applying appropriate fermentation techniques, agro-waste can be used in cost-effective production of antibiotics. Recent studies reported the production of neomycin, oxytetracycline, and rifamycin using agro-wastes as substrate by solid state fermentation (SoSF). Several microorganisms were used for the production of these valuable products. In addition, the external energy sources were supplied to enhance the production of antibiotic.KeywordsAgro-wasteAntibioticsSolid state fermentation (SoSF)Mechanism of actionProcess optimization
... To enrich the hydrogen producers in the sludge, methanogens need to be eliminated from the inoculum and that is where the aid of seed pretreatment is required. There are various types of seed pretreatment methods: chemical treatment, heat-shock treatment, and organic load-shock treatment (O-Thong et al. 2009). Chemical treatments involve either acid or alkali to eliminate methanogens at extreme pH. ...
Biohydrogen is a clean fuel, which can be produced through direct or indirect biophotolysis, photo-fermentation, and dark fermentation by microorganisms. In dark fermentation, organic waste degradation and hydrogen production go simultaneously. A variety of substrates from industrial wastewater to agricultural solid wastes have been used for biohydrogen production. Obligate anaerobes from genera Clostridium and Desulfovibrio species and facultative anaerobes from Citrobacter, Enterobacter, Klebsiella, and Bacillus species are known to utilize the organic wastes to produce hydrogen through dark fermentation. Biohydrogen production in batch and fed-batch reactors at lab-scale to pilot scale have been demonstrated by several researchers. The major bottlenecks for the large-scale production of biohydrogen are the costs of plant establishment and maintenance. This study gives an overview of the potential microbes and technology involved in the biohydrogen production from organic wastes through dark fermentation and the factors to be addressed for its commercial production.KeywordsBiohydrogenBottlenecksCommercializationDark fermentation
... Additionally, different methods of pretreatment of the inoculums have been used for hydrogen production enhancement by releasing the organics to liquid and decrease the hydrogen consumers. These include mechanical treatment (milling, grinding, and lysis-centrifugation), thermal treatment [15], acid treatment [16], alkali treatment [17], acid and alkali treatment [18], chemical treatment (Bromoethane sulfonic acid) [19][20][21], and heat-alkaline treatment [22,23]. Furthermore, our previous study showed that both yield and concentration of H 2 were the highest at pH 7 treated by chemical treatment (CT), which were 4.44 mL/g SCOD and 280.82 mL/L, while yield and concentration of the VFAs were the highest at pH 6 treated by chemical treatment (CT), which were 926.21 mg/g SCOD and 55.44 g/L, respectively [24]. ...
Food waste (FW) contains high amounts of organic substances and moisture like lipids, starches, and proteins. Dark fermentation (DF) has the ability to produce from FW high-value by-products, like lactic acid (LA), hydrogen (H2), alcohols (EtOH), short-chain fatty acids, and methane which produced in the oxidative stage of anaerobic digestion. Moreover, it has been proved that hydrogen is one of the promising energy sources which is vital for shrinking dependency on fossil fuels. Otherwise, volatile fatty acids (VFAs) have a wide-ranging of applications such as the utilization of an alternative carbon source. The comparison between the trial PHP (pretreatment, HRT, and pH) and the control system at the same hydraulic retention time (HRT) levels was studied. The paired-samples t-test showed that the trial system has a highly significant difference compared to the control system (P < 0.0001( for both H2 and VFAs production, where H2 production rate was 0.19 mL/L at the control system, and 263.82 mL/L at the trial system. On the other hand, the trial was 1.8 times higher than the control system for VFAs production. Based on the obtained results, the trial system is recommended for producing H2 and VFAs from waste food.
... Enhancement of microbial communities through pretreatment of the inoculum is an effective way to induce changes in the communities to improve process performance. This approach has already been successfully tested, but mainly for optimizing the acidogenic step in H 2 production through dark fermentation [21][22][23]. Pretreatments used in those studies consisted of modifications of the pH and temperature applied to the inoculum. Since microbial communities involved in acidogenic processes could use H 2 as an electron donor to shift their metabolism to produce alcohols [1,5,24], the same inoculum pretreatments could be applied to improve solventogenic processes. ...
This work reports the effect of four different physicochemical pretreatments (acidic, thermal, acidic-thermal, and thermal-acidic) on an anaerobic inoculum aiming at alcohols production, using acetate and butyrate as carbon sources and hydrogen as co-substrate. Pretreatments were carried out to select microbial communities more able to use hydrogen to metabolize volatile fatty acids into their respective alcohols. Experiments were conducted in single batches using acetate and butyrate as substrates at 30 °C and with a pressurized headspace of pure H2 at 2.15 atm (218.2 MPa). Thermal and acidic-thermal pretreatments lead to higher production of both ethanol and butanol, indicating that these pretreatments successfully selected communities more suitable for acetate and butyrate solventogenesis. Kinetics modelling shows that the highest attainable concentrations of ethanol and butanol produced were 122 mg L⁻¹ and 97 mg L⁻¹ for the thermal pretreatment (after 17.5 days) and 87 mg L⁻¹ and 143 mg L⁻¹ for the acidic-thermal pretreatment (after 18.9 days). Process thermodynamics indicated that high H2 partial pressure favoured solventogenic metabolic pathways. Sequencing data showed that both thermal and acidic-thermal pretreatments selected mainly the bacterial genera Pseudomonas, Brevundimonas, and Clostridium. Acidic-thermal pretreatment selected a bacterial community more adapted to the conversion of acetate and butyrate into ethanol and butanol, respectively. Thermal-acidic pretreatment was unstable, showing significant variability between replicates. Acidic pretreatment showed the lowest alcohol production.
... Enhancement of microbial communities through pretreatment of the inoculum is an effective way to induce changes in the communities to improve process performance. This approach has already been successfully tested, but mainly for optimizing the acidogenic step in H 2 production through dark fermentation [21][22][23]. ...
Four different physicochemical pretreatments on an anaerobic inoculum used for alcohol production from acetate and butyrate are evaluated. Experiments were conducted in single batches using acetate and butyrate as substrates at 30°C and with a pressurized headspace of pure H 2 at 2.15 atm (218.2 MPa). Thermal and acidic-thermal pretreatments lead to higher production of both ethanol and butanol. Modelling shows that the highest attainable concentrations of ethanol and butanol produced were 122 mg L ⁻¹ and 97 mg L ⁻¹ for the thermal pretreatment (after 17.5 days) and 87 mg L ⁻¹ and 143 mg L ⁻¹ for the acidic-thermal pretreatment (after 18.9 days). Thermodynamic data indicated that a high H 2 partial pressure favoured solventogenic metabolic pathways. Acidic-thermal pretreatment selected a bacterial community more adapted to the conversion of acetate and butyrate into ethanol and butanol, respectively. Thermal-acidic pretreatment was unstable, showing significant variability between replicates. Acidic pretreatment showed the lowest alcohol production.
... Enhancement of microbial communities through pretreatment of the inoculum is an effective way to induce changes in the communities that will improve process performance. This approach has already been successfully tested, but mainly for the optimization of the acidogenic step in H 2 production through anaerobic dark fermentation [25][26][27][28][29][30][31][32][33][34][35]. Pretreatments used in those studies consisted in modifications of the pH and temperature applied to the inoculum. ...
This work evaluates four different physicochemical pretreatments (acidic, thermal, acidic-thermal and thermal-acidic) on an anaerobic inoculum used for alcohol production from acetate and butyrate. All experiments were conducted in single batches using acetate and butyrate as substrates at 30°C and with a pressurized headspace of pure H 2 at 2.15 atm (218.2 MPa). Thermal and acidic-thermal pretreatments lead to higher production of both ethanol and butanol. Mathematical modelling shows that the highest attainable concentrations of ethanol and butanol produced were 122 mg L ⁻¹ and 97 mg L ⁻¹ for the thermal pretreatment (after 17.5 days) and 87 mg L ⁻¹ and 143 mg L ⁻¹ for the acidic-thermal pretreatment (after 18.9 days). Acetate was produced in all assays. Thermodynamic data indicated that a high H 2 partial pressure favoured solventogenic metabolic pathways. Finally, sequencing data showed that both thermal and acidic-thermal pretreatments selected mainly the bacterial genera Pseudomonas , Brevundimonas and Clostridium . The acidic-thermal pretreatment selected a bacterial community more adapted to the conversion of acetate and butyrate into ethanol and butanol, respectively. Thermal-acidic pretreatment was unstable, showing significant variability between replicates. Acidic pretreatment showed the lowest alcohol production.
... The Lactobacillus genus most likely mediated the high lactic activity observed in the FDC, whilst the Thermoanaerobacterium genus was most likely associated to the production of HBu and HAc, both measured as secondary metabolites (Fig. 4). The Thermoanaerobacterium genus has often been pointed out as playing a determining role in bioH 2 production in the (hyper)thermophilic fermentation of different sugarcane-derived substrates, such as vinasse [18,20,58,65,71], bagasse [72] and molasses [33,34], commonly producing HAc, HBu, HLa and succinic acid as the soluble phase metabolites [18,71,73,74]. A relatively high RA (7.41%) was also observed for this genus in the bed region of the AnSTBR at the end of the operation (HRT = 10.0 hcondition II, S6; Fig. 6), most likely using HLa as the precursor for both HBu and bioH 2 . ...
Using pure sugars for fermentative biohydrogen (bioH2) production is known as an economically impeditive approach due to the high costs of raw materials, which leads to the frequent exploitation of residual streams. However, the potentials of using biodigestion as a core processing step in sugarcane biorefineries opens up a wide range of biotechnological possibilities, in which sugar-rich materials may be fermented without adding acquisition costs. This study aimed to finalize the full optimization of the continuous long-term (630 d) thermophilic (55°C) bioH2 production from molasses, defining adequate hydraulic retention time (HRT) levels. Details of temporal and spatial metabolite distribution profiles coupled to the characterization of microbial communities provided the most complete picture of molasses acidogenesis to date. BioH2 evolution (8.5 NL-H2 L⁻¹ d⁻¹) in full optimized conditions, i.e., HRT ≈ 10.0 h, organic loading rate (OLR) ≈ 86.0 kg-CODt m⁻³ d⁻¹ and pH ≈ 5.38, exceeded the individual application of optimal OLR (4.5 NL-H2 L⁻¹ d⁻¹) and pH (2.4 NL-H2 L⁻¹ d⁻¹) by almost 200% and 350%, respectively. Biomass washout naturally controlled substrate availability (6.21 ± 2.1 g-COD g⁻¹VSS d⁻¹ for over 300 d), preventing performance losses in the long-term. BioH2 derived from acetic- and butyric-type fermentations was observed in the basal portion of the reactor, whilst butyrate and bioH2 from reverse β-oxidation using lactate prevailed in the bed region. The Thermoanaerobacterium genus was the main group involved in such pathways, and the contribution of the Caproiciproducens genus in caproate production was also revealed. These findings provide consistent directions to scale-up acidogenic systems towards an effective exploitation of full-scale bioH2 production.
... Among these, thermally pre-treated substrate produced 1.5e4 times higher hydrogen yield. Studies also denoted that it would lead to complete the inhibition of hydrogen consuming microbes, which make the process uneconomical [188,189]. Cui et al. [190], pre-treated leaf waste with enzymatic technique where maximum hydrogen yield was achieved as 44.92 mL per gram dry poplar leaves, which was 3 times higher than untreated as well as 1.34 times higher than acidic pre-treated substrate. Contreras-D avila et al. [191] studied enzymatic hydrolysis of Agave tequilana bagasse both continuous stirred tank reactor and a trickling bed reactor. ...
... Among these, thermally pre-treated substrate produced 1.5e4 times higher hydrogen yield. Studies also denoted that it would lead to complete the inhibition of hydrogen consuming microbes, which make the process uneconomical [188,189]. Cui et al. [190], pre-treated leaf waste with enzymatic technique where maximum hydrogen yield was achieved as 44.92 mL per gram dry poplar leaves, which was 3 times higher than untreated as well as 1.34 times higher than acidic pre-treated substrate. Contreras-D avila et al. [191] studied enzymatic hydrolysis of Agave tequilana bagasse both continuous stirred tank reactor and a trickling bed reactor. ...
The potential to operate energy efficient and less expensive production methods are important in biohydrogen production. Biological hydrogen production is often constrained by less productivity. However, to obtain industrial level implementation, greater productivity is essential. Researches on various bioreactors configurations and influencing factors were deeply investigated in this regard. The bioreactors operated in batch mode are appropriate for preliminary optimization whereas industrial level execution needs continuous mode. The main objective of this review is to recap the limitations and constraints associated with bioreactor operation and to list out the enhancement approaches that are currently investigated for improved biohydrogen generation. Recent approaches designed towards biohydrogen production enhancement such as substrate pre-treatments, inhibitors removal, bioaugmentation, immobilization, effluent recycling, buffering capacity maintenance, exploitation of by-products etc., are reviewed thoroughly.
... Typically, the use of anaerobic sludge and granules requires an additional step of pretreatment to inhibit the methanogens. Several pretreatment methods have been reported in the literature, including heat-shock, load-shock, the use of chemical such as acid, alkali, and 2-bromoethanesulfonate acid (BESA), aeration, and electrical pretreatment (O-Thong et al., 2009;Pachapur et al., 2019). Among these, heat treatment is the most popularly used as it is simple and inexpensive (Li & Fang, 2007). ...
Third generation biomass, i.e. microalgae, has emerged as a promising alternative to first and second generation biomass for biohydrogen production. However, its utilization is still low at present, due to several reasons including the strong and rigidity of the microalgal cell wall that limit the hydrolysis efficiency during dark fermentation (DF) and photofermentation (PF) processes. To improve the utilization efficiency of microalgal biomass, it is crucial that important aspects related to the production of the biomass and the following processes are elaborated. Thus, this article provides detailed overview of algal strains, cultivation, and harvesting. It also presents recent research and detailed information on microalgal biomass pretreatment, and biohydrogen production through DF, PF, and co-digestion of microalgal biomass with organic materials. Furthermore, factors affecting fermentation processes performance and the use of molecular techniques in biohydrogen production are presented. This review also discusses challenges and future prospects towards biohydrogen production from microalgal biomass.
... O-Thong et al., 2009 glu a = glucose, xyl b = xylose, NA = data not available Chapter 7: General discussion and conclusion ...
The aim of this thesis was to enhance thermophilic dark fermentative hydrogen production by using microbial strategies (bioaugmentation and synthetic co-cultures) and by increasing the understanding on the microbial community dynamics especially during stress conditions such as fluctuating temperatures and elevated substrate concentrations. To study the effects of sudden short-term temperature fluctuations, batch cultures initially incubated at 55°C (control) were subjected to downward (from 55°C to 35°C or 45°C) or upward (from 55°C to 65°C or 75°C) temperature shifts for 48 hours after which they were incubated again at 55°C for two consecutive batch cycles. The results showed that sudden, temporal upward and downward temperature fluctuations had a direct impact on the hydrogen yield as well as the microbial community structure. Cultures exposed to downward temperature fluctuation recovered more rapidly enabling almost similar hydrogen yield (92-96%) as the control culture kept at 55 °C. On the contrary, upward temperature shifts from 55 to 65 or 75 °C had more significant negative effect on dark fermentative hydrogen production as the yield remained significantly lower (54-79%) for the exposed cultures compared to the control culture. To improve the stability of hydrogen production during temperature fluctuations and to speed up the recovery, mixed microbial consortium undergoing a period of either downward or upward temperature fluctuation was augmented with a synthetic mix culture containing well-known hydrogen producers. The addition of new species into the native consortium significantly improved hydrogen production both during and after the fluctuations. However, when the bioaugmentation was applied during the temperature fluctuation, hydrogen production was enhanced. This study also investigated the dynamics between pure cultures and co-cultures of highly specialized hydrogen producers, Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana. The highest hydrogen yield (2.8 ± 0.1 mol H2 mol-1 glucose) was obtained with a synthetic co-culture which resulted in a 3.3 or 12% increase in hydrogen yield when compared to pure cultures of C. saccharolyticus or T. neapolitana, respectively. Furthermore, quantitative polymerase chain reaction (qPCR) based method for monitoring the growth and contribution of T. neapolitana in synthetic co-cultures was developed. With this method, it was verified that T. neapolitana was an active member of the synthetic co-culture. The effect of different feed glucose concentrations (from 5.6 to 111.0 mmol L-1) on hydrogen production was investigated with and without augmenting the culture with T. neapolitana. Compared to the control (without T. neapolitana), bioaugmentated culture resulted in higher hydrogen yields in almost all the concentrations studied even though hydrogen yield decreased the feed glucose concentration was increased. The presence of T. neapolitana also had a significant impact on the metabolite distribution when compared to the control.In summary, this study showed that thermophilic dark fermentative hydrogen production can be enhanced by using synthetic co-cultures or bioaugmentation. The highest hydrogen yield in this study was obtained with the synthetic co-culture, although it should be considered that the incubation conditions differed from those used for the mixed cultures in this study. The use of molecular methods such as qPCR and high-throughput sequencing also helped to understand the role of certain species in the microbial consortia and improved the understanding of the microbial community dynamics during stress conditions
... Different methods such as acid, base, heating, using chemical compound, aeration, and ultrasonication were used as a pretreatment for enriching biohydrogen-producing bacteria. [4][5][6][7] The method that was used for sludge pretreatment is an effective factor in the dominance of special pathway for biohydrogen production, for example, by heating sludge as a pretreatment method can select spore-forming bacteria such as clostridia or pretreatment by aeration led to Clostridium sp. and Enterobacter dominance. [6,8] The efficiency of biohydrogen production process is different depending on pretreatment method, dominance bacteria, substrate, and metabolic pathway. ...
... Treating the culture with acid (pH 3) or alkaline (pH 9 to 11.5) enriches H 2 -producing bacteria in anaerobic mixed cultures [226][227][228]. Acid/base treatment of cultures in industrial-scale bioreactors by maintaining pH 3 or pH 9 to 11.5 would consume large quantities of acid and alkaline and requires equipment for feeding, mixing, pH control, and then neutralizing or adjusting the pH to the desired initial pH (usually 5.0-5.5). ...
Industrial organic waste from food processing, livestock production, brewery, bakery, and other related industries is a renewable substrate for anaerobic digestion to produce methane (CH4) or with some process manipulation and control to produce hydrogen (H2). Type of waste, its strength, presence of any toxic compounds, and other specific characteristics affect the operating conditions such as organic loading rate, hydraulic retention time, substrate pretreatment, as well as the yield and the rate of H2 production from industrial waste. Therefore, they need to be optimized for each waste. Research is required on the modeling, cost analysis, economic evaluation, comparative studies about the effect of bioreactor design, as well as on combining several industrial wastes to prepare a well-balanced substrate for H2-producing mixed culture dark fermentation.
... They reported that the fermentation type depended on the inoculum pre-treatment (Ren et al., 2008) or on pH (Khanal et al., ). Similarly to our study, the heat-shock pretreatment is commonly considered as the most suitable method to enrich hydrogen producing bacteria and eliminate non-spore forming bacteria such as methanogens (Mu et al., 2007;Wang and Wan, 2008;O-Thong et al., 2009). After treatment, the spore-forming bacteria are mainly belonging to the genus Clostridium sp. ...
This study aims to investigate the effect of solid substrates composition on hydrogen production performances, metabolic pathways and microbial community changes in batch reactor and their dynamics in continuous reactors (CSTR). Hydrogen is an ideal energy carrier which has gained scientific interest over the past decade. Biological H2, so-called biohydrogen, can especially be produced by dark fermentation processes concomitantly with value-added molecules (i.e. metabolic end-products), while organic waste is treated. However, the effect of solid organic waste composition on biohydrogen production in dark fermentation has not yet been clearly elucidated. In this study, a bibliographic review was made on hydrogen production from agricultural waste. This survey on literature showed that diverse performances were reported on hydrogen production due to the variability in substrate compositions and experimental conditions. After having optimized a protocol of biohydrogen potential test (BHP), a wide variety of organic solid substrates aiming to covering a large range of solid waste was tested to provide a comparable data analysis. The results of a PLS regression showed that only soluble carbohydrates or easily available carbohydrates correlated with hydrogen production. Furthermore, hydrogen yields correlated as well with butyrate H2-producing pathway which is consistent with the literature knowledge. A predictive model of hydrogen yield according to carbohydrate content was proposed. Then, experiments were carried out in CSTR with Jerusalem artichoke tubers as a case study. It was shown that low organic loading rate favored continuous hydrogen production while higher organic loading introduced hydrogen competition pathways and decreased the overall hydrogen yields. Moereover, 16S rRNA gene based CE-SSCP profiles showed that increasing OLR had a significant effect on the microbial diversity by favoring the implementation of microorganisms not producing hydrogen, i.e. lactic acid bacteria
... Intuitively, it is believed that higher temperatures require shorter exposure times, however the best combination to improve H2 yields is strongly dependent on the inoculum source, i.e. native microbial diversity. Nonetheless, 2.49 molH2mol -1 hexose is the highest H2 yield obtained using heat pre-treatment, in this case from anaerobic sludge treated at 100 °C for 60 min, using sucrose as substrate [22], [64]- [66]. (Adapted from [22]) ...
Dark fermentation (DF) is a biological process used to produce hydrogen (H2). In this process a wide variety of substrates could be used including simple substrates such as glucose or more complex substrates such as industrial wastewaters and waste. Mixed cultures can be used as inoculum that are more robust than pure cultures. However, H2 producers and consumers can coexist in such mixed cultures, so that they often have to be pre-treated to inhibit H2-consumers. Moreover, operational parameters such as pH and temperature play a key role in the selection of H2-producing bacterial community. Up to date, great efforts have been made to optimise the operational parameters, but only few controllers are available to maintain the DF process stable. In this context, an electro-fermentation (EF) process is proposed as a new tool to control bioprocesses through polarised electrodes. Depending on the applied potential, EF can occur at the anode or cathode, acting either as electron sink or additional source of energy, respectively. High current densities are not necessary to have a significant impact on cell metabolism since the electrical current is not the main electrons source, nor the product of interest. The mechanisms behind EF are still unknown, but microbial interactions between fermentative and electroactive bacteria may be the key factor of the process. The objective of this thesis was: "Better understanding of the EF mechanisms through the characterization of microbial interspecies-interactions as well as interactions with the polarized electrode". Our results show that the presence of polarized electrodes led to the selection of H2-producing bacteria, and more particularly from Enterobacteriaceae and Clostridiaceae families. Such microbial selection was concomitant with a significant increase in H2 and butyrate production, at the expense of lactate production. However, when different inoculum were used, different behaviours were observed with an increase, a decreased or no effect on H2 production. This observation evidences that the inoculum microbial community composition, and more particularly the relative abundance of the Clostridiaceae family, can significantly affect the microbial community behaviour in EF, i.e. microbial community trajectories and the related metabolic patterns. Finally, the microbial interactions were further investigated with a mixed inoculum enriched in G. sulfurreducens, as well-known electroactive bacteria. Here, a substantial change in the metabolic pathways towards higher H2 and butyrate production was observed, at the expense of 2,3-butanediol production. This change was associated with an increase in relative abundance of the Clostridiaceae family at the end of fermentation, probably due to a cooperative growth that G. sulfurreducens occurring with the members of the Clostridiaceae family. Overall, although the mechanisms behind the microbial interactions are not yet well know, the EF process showed a great potential as a new type of control for mixed-culture bioprocesses with significant effects of the polarized electrodes on glucose fermentation.
... The inhibition of methanogenesis to generate intermediate products is already widely used in experiments for hydrogen production. Some of the techniques used to block the process are: low hydraulic retention time (HRT) for preventing the growth of methanogens (Thanwised et al., 2012), pre-treatment, such as heat-shock (Leaño and Babel, 2012), acid shock (Leite, 2008) and alkaline shock (O-Thong et al., 2009) of the inoculum sludge for eliminating methanogens, and two-stage reactor (acidogenic/methanogenic) for inhibiting methanogenesis in the acidogenic reactor due to low pH (Luo et al., 2011) or chemical inhibition (Bouwer and McCarty, 1983). ...
Acidogenic fermentation (AF) of cassava wastewater was investigated with adapted and unadapted biomass with and without methanogenic inhibition techniques to enhance accumulation of volatile fatty acids (VFA) in batch tests. Subsequently, the possibility of VFA chain elongation for the production of medium-chain fatty acids (MCFA) by means of ethanol addition to the fermentation broth was evaluated. With unadapted sludge, 84.1% and 66.1% acidification could be obtained for thermally inhibited and non-inhibited conditions respectively, results 10% and 45% higher than the degrees of acidification obtained under the same conditions with adapted sludge. Results show that neither sludge adaptation nor heat treatment but rather the pH of the fermentation broth was most relevant for enhancing VFA production, the optimum pH range being 5.5 to 6. The main product was butyric acid (87% of total acid produced), and addition of ethanol to the fermentation broth promoted MCFA formation.
... The inhibition of methanogenesis to generate intermediate products is already widely used in experiments for hydrogen production. Some of the techniques used to block the process are: low hydraulic retention time (HRT) for preventing the growth of methanogens (Thanwised et al., 2012), pre-treatment, such as heat-shock (Leaño and Babel, 2012), acid shock (Leite, 2008) and alkaline shock (O-Thong et al., 2009) of the inoculum sludge for eliminating methanogens, and two-stage reactor (acidogenic/methanogenic) for inhibiting methanogenesis in the acidogenic reactor due to low pH (Luo et al., 2011) or chemical inhibition (Bouwer and McCarty, 1983). ...
Acidogenic fermentation (AF) of cassava wastewater was investigated with adapted and unadapted biomass with and without methanogenic inhibition techniques to enhance accumulation of volatile fatty acids (VFA) in batch tests. Subsequently, the possibility of VFA chain elongation for the production of medium-chain fatty acids (MCFA) by means of ethanol addition to the fermentation broth was evaluated. With unadapted sludge, 84.1% and 66.1% acidification could be obtained for thermally inhibited and non-inhibited conditions respectively, results 10% and 45% higher than the degrees of acidification obtained under the same conditions with adapted sludge. Results show that neither sludge adaptation nor heat treatment but rather the pH of the fermentation broth was most relevant for enhancing VFA production, the optimum pH range being 5.5 to 6. The main product was butyric acid (87% of total acid produced), and addition of ethanol to the fermentation broth promoted MCFA formation.
... The mesophilic range is the most used; some of the most remarkable hydrogen-producing bacteria (Clostridium, Enterobacter, and Bacillus) (see Fig. 4.1) are active at this temperature range [15,67]. On the other hand, gases are less soluble at higher temperatures and thermophilic HPB, belonging to the Thermoanaerobacterium genus, can produce up to 1.96 mol H 2 mol À1 hexose at 60°C after 48 h [68]. Thus, high-temperature sidestreams from food-processing industries, such as thermomechanical pulping wastewaters that release at 50-80°C, have been subjected to dark fermentation at 70°C, using the heat produced by the plant. ...
... These can be restricted by load shock treatment, i.e., to highly enrich the environment with hydrogen producing in ocula so that they dominate the methanogens. This is quite effective for increasing the productivity of biohydrogen (Sompong et al. 2009). Aeration and ultrasonication can also be used effectively to suppress hydrogen consumers (Kotay and Das 2008). ...
The desire to reach beyond fossil fuels for energy sources has prompted the exploration of various unconventional energy sources. Production of hydrogen using various groups of microbes (archaea, bacteria, cyanobacteria, fungi, and algae) is one potential alternative of traditional fossil fuel. Among different microbes, cyano bacteria are highly promising for hydrogen production. In comparison to the traditional ways of hydrogen production (chemical), microbial hydrogen production is commercially viable and eco‐friendly. Hydrogen may be produced by several processes, including electrolysis of water, thermocatalytic reformation of hydrogen‐rich organic compounds, and biological processes. Biological productions of hydrogen (biohydrogen) technologies provide a wide range of approaches to generate hydrogen, including direct biophotolysis, indirect biophotolysis, photo‐fermentations, dark‐fermentation, and microbial electrolysis cells (MECs). This chapter highlights the basics of microbial hydrogen production, microbial abundance involved, large‐scale hydrogen production, and its prospects.
Dans le contexte actuel de transition énergétique, la perspective de production d’un vecteur énergétique comme l’hydrogène via un procédé biologique (fermentation obscure) présente un intérêt majeur. Dans cette thèse, des modèles mathématiques ont été développés pour modéliser et optimiser le procédé de production d’hydrogène par fermentation, dans l’objectif de transférer ce processus de l’échelle laboratoire à l’échelle pilote. Une optimisation de la teneur initiale en substrat modèle pour obtenir un rendement d’hydrogène élevé a été réalisée en se basant sur un modèle mathématique modifié, intégrant un facteur de correction permettant de modéliser toutes les phases de fermentation. Une vitesse de production d’hydrogène de 5 L/Lbioréacteur/j a été obtenue à une concentration en substrat de 13 g/L. Une étude des cinétiques microbiennes lors la production d’hydrogène par fermentation endogène d’une biomasse viticole a été réalisée en identifiant les interactions ayant lieu entre les bactéries de différents genres ainsi qu’en optimisant la charge initiale de biomasse. Enfin, l’application du plan d’expériences de Plackett-Burman a permis d’augmenter la performance de production d’hydrogène par fermentation d’une biomasse caféicole jusqu’à 48% en conjuguant les prétraitements enzymatique, thermique et chimique.
In recent years, the demand for high-quality biofuels from renewable sources has become an aspirational goal to offer a clean environment by alternating the depleting fossil fuels to meet future energy needs. In this aspect, biohythane production from wastes has received extensive research interest since it contains superior fuel characteristics than the promising conventional biofuel i.e. biogas. The main aim is to promote research and potentials of biohythane production by a systematic review of scientific literature on the biohythane production pathways, substrate/microbial consortium suitability, reactor design, and influential process/operational factors. Reactor configuration also decides the product yield in addition to other key factors like waste composition, temperature, pH, retention time and loading rates. Hence, a detailed emphasis on different reactor configurations with respect to the type of feedstock has also been given. The technical challenges are highlighted towards process optimization and system scale up. Meanwhile, solutions to improve product yield, technoeconomics, applications and key policy and governance factors to build a hydrogen based society have also been discussed.
Hydrogen is an energy carrier with a very high energy density (>119 MJ/kg). Pure hydrogen is barely available; thus, it requires extraction from its compounds. Steam reforming and water electrolysis are commercially viable technologies for hydrogen production from water, alcohols, methane, and other hydrocarbons; however, both processes are energy-intensive. Current study aims at understanding the methane and ethanol-water mixture pathway to generate hydrogen molecules. The various intermediate species (like CHX, CH2O, CH3CHO) are generated before decomposing methane/ethanol into hydrogen radicals, which later combine to form hydrogen molecules. The study further discusses the various operating parameters involved in plasma reforming reactors. All the reactors work on the same principle, generating plasma to excite electrons for collision. The dielectric barrier discharge reactor can be operated with or without a catalyst; however, feed flow rate and discharge power are the most influencing parameters. In a pulsed plasma reactor, feed flow rate, electrode velocity, and gap are the main factors that can raise methane conversion (40–60%). While the gliding arc plasma reactor can generate up to 50% hydrogen yield at optimized values of oxygen/carbon ratio and residence time, the hydrogen yield in the microwave plasma reactor is affected by flow rate and feed concentration. Therefore, all the reactors have the potential to generate hydrogen at lower energy demand.
This review mainly determines novel and advance physical, chemical, physico–chemical, microbiological and nanotechnology–based pretreatment techniques in lignocellulosic biomass pretreatment for bio–H2 production. Further, aim of this review is to gain the knowledge on the lignocellulosic biomass pretreatment and its priority on the efficacy of bio–H2 and positive findings. The influence of various pretreatment techniques on the structure of lignocellulosic biomass have presented with the pros and cons, especially about the cellulose digestibility and the interference by generation of inhibitory compounds in the bio–enzymatic technique as such compounds is toxic. The result implies that the stepwise pretreatment technique only can ensure eventually the lignocellulosic biomass materials fermentation to yield bio–H2. Though, the mentioned pretreatment steps are still a challenge to procure cost–effective large–scale conversion of lignocellulosic biomass into fermentable sugars along with low inhibitory concentration.
Anaerobic digestion serves as a feasible and practical technology that converts food waste into biogas. Effective food waste-digesting inoculum must be capable of handling the heterogeneity of organic fractions. Therefore, the preparation of such an inoculum can be challenging. In this work, we explored a new approach to prepare the inoculum from palm oil mill effluent (POME), which is an alternative source of microbial consortium abundantly available in tropical countries, to be a food waste-digesting inoculum. By employing POME as an initial inoculum, our acclimatization was divided into three strategies, which were low, medium, and high-rate acclimatization. The result showed that low acclimatization was a successful strategy to adapt to the consuming behavior of microorganisms inside the anaerobic digestion system. The low acclimatization could provide a methane yield (513.90 mL-CH4/g-volatile solidadded) and a methane production rate (1.02 mL-CH4/g-volatile solidadded.h) higher than those in other reports by up to 12% and 80% for the methane yield and methane production rate, respectively. In addition, we also explored changes in microbial communities throughout the process. Microbial communities significantly shifted from lignocellulose-digesting bacteria to protein, lipid, and carbohydrate-digesting bacteria. Particularly, Papillibacter, Ruminiclostridium, Ruminiclostridium 1, and Ruminococcaceae UCG-010 genera were shifted to Proteiniphilu, Christensenellaceae R-7 group, and W5 genera. Interestingly, the major populations of methane-producing microbes changed from Methanosarcina to Methanosaeta. In low acetate environments, such as those found in stable AD systems, Methanosaeta should outnumber Methanosarcina. The presence of dominating Methanosaeta in the system showed the strategy's stability. The acclimatization strategy developed in this study exhibited promising potential to be used as an alternative method for waste management as well as a sustainable energy source due to its efficient methane gas production using food waste as the substrate.
The two-stage anaerobic digestion (AD) is gaining popularity because of the process stability and possibility of recovering multiple-resources such as biohydrogen and organic acids from the first stage dark fermentation (DF) and methane in the AD as the second stage while treating the organic waste. As the performance of two-stage processes is influenced by the type of substrate and operational conditions, there have been several experiments at laboratory and pilot scales to determine the optimum conditions. The main objective of this review is to provide an updated overview of advancements in biohythane and organic acids production from food waste (FW) in the two-stage DF-AD process. Likewise, this work also provides an insight into the economic and future prospective of utilizing organic acids for different biochemicals such as polyhydroxyal-kanoates, polylactate, and microalgal biomass production. The integration of optimum operational parameters, pretreatment methods, types of bioreactors is essential in combined DF-AD processes. The parameters and reactor configuration have to be optimized depending upon the targeted end-products. More research into the techno-economic analysis of different bio-reactor configurations for long-term operations in an integrated DF-AD process with FW as a feedstock is needed to realize its viability for commercial application. Graphical abstract
Residual Fermented Solid (RFS) is the used biocatalyst obtained after enzymatic biodiesel production carried out applying the fermented solid (FS) with lipase activity. Approximately 350 g of RFS are generated for each liter of biodiesel produced from palm residues fermented solid. In this study, this residue was used for the first time as a raw material for biological hydrogen production through dark fermentation and sequential application of the hydrogen production liquid waste (HPLW) for methane obtainment via anaerobic digestion. The RFS was composed mostly of oils and fats (60% wt.%), and carbohydrates, such as mannose, glucose, and xylose. Hydrogen yield reached 239 ± 44 mL H2/L after 24 h of fermentation using 31 gRFS/L at the beginning of the process. Additionally, 204 ± 13 mL CH4/g COD were produced through the anaerobic digestion of HPLW, which represented 61% of efficiency.
Water utilities generate enormous amounts of sewage sludge globally each year. This chapter introduces the characteristics of sewage sludge and anaerobic sludge digestion. The state‐of‐the‐art technologies for enhancing methane and hydrogen productions from sewage sludge are also elucidated, including physical, chemical, and biological pretreatment. Emphasis is put on their effect on methane and hydrogen production performance, with an increase of 10–340% in methane production and an increase of 20–1300% in hydrogen production. In general, thermal pretreatment, free nitrous acid pretreatment, free ammonia pretreatment, and temperature‐phased anaerobic digestion show advantages over the other pretreatment technologies. In addition, ultrasonic pretreatment (<4400 kJ/kg total solids) will also be promising if pathogen destruction is not a main concern. In the future, various pretreatment technologies should be implemented to the same sludge source in order to avoid the bias imposed by the different sludge sources.
Hydrogen is considered the energy of the future. In order to meet the requirements of a green production, many technological advances were achieved in the last 20 years. Reviewing these advances allows the understanding the constraints of the sector and the identification of the paths for future research. In this work three routes for renewable hydrogen production from renewable sources were reviewed with focus on patented technologies: dissociation of the H2O molecule, microbial production and thermochemical processes. Three patent databases were accessed, covering documents written in English, Spanish and Portuguese. Water dissociation is the only commercial technology and most efforts were identified in new materials for electrodes, new catalysts and reagent recycling. Microbial production is thermodynamic limited but strategies to maximize the production and overcome economic barriers are mostly related to the use of wastewaters, improving microbial communities' composition and genetic manipulation. Most patents of thermochemical production patents brought contributions to the efficient use of energy especially by the combination with other energy producing technologies. The establishment of a green hydrogen society is strongly dependent of future developments on new materials, recycle of chemical catalysts and efficient use of energy in the short-term.
The environmental impacts associated with fossil fuels and depletion of their resources motivated the development of alternate fuels. Hydrogen is an attractive alternate fuel with high heating value and no environmental impact. Although hydrogen is majorly produced by steam reforming, biochemical production especially from carbohydrate rich wastes is of great interest. Hydrogen production from these wastes is carried out by indirect/direct biophotolysis, dark fermentation, two‐stage fermentation and photofermentation. The major constraints of these processes are low hydrogen evolution rate and less yield at large scale. However, effective pretreatment of substrates and inoculum maximize the yield of hydrogen. The factors influence the hydrogen production include nature of microorganism, biochemical process/reactor, temperature, pH, ionic strength, hydraulic retention time, hydrogen and carbon dioxide partial pressure, organic acid concentration, and C/N ratio. The commercial exploitation of biohydrogen production is hindered by lack of high potential microorganism and bioreactor. Hence, it demands multidisciplinary research to understand the fundamental underlying principles besides the development of microbial strains for industrial applications.
The following study examines the potential of process liquor from hydrothermal
carbonization of bio wastes to produce hydrogen gas in an anaerobic fermentation
process. In comparison to other studies, the aim was not to produce methane, but
hydrogen instead. For suppression of methanogenic Bacteria, heat treatment of
digested sludge was considered a possibility.
Experiments carried out with heat treated sludge yield no results. No gas production
was observable.
Heat-treatment wasn’t used in further experiments. A temperature of 60 °C was set
as the sole condition to suppress methanogenic activity. The process liquor was used
diluted and in pure form. Additionally, molasses was used in some experiments as a
co-substrate.
The results show, that no hydrogen was produced from process liquor without the
use of molasses. Methanogenic activity was not suppressed, by only using a
temperature of 60 °C. Methane was produced with yields ranging from
125 NL CH4 kg-1 VS to 177 NL CH4 kg-1 VS.
Waste organic biomass is regarded as the most suitable renewable source for conversion to produce biofuels and biochemicals. Owing to its high‐energy potential and abundancy, lignocellulosic biomass can be utilized to produce alternative energy in the form of gaseous and liquid biofuels. Microbial conversion of waste biomass is found to be the most successful technology for the generation of biohydrogen through dark fermentation. Hydrogen is considered as the promising renewable green energy source for a sustainable future. Different biological hydrogen production technologies along with process parameters are described in this review paper although the emphasis is on dark fermentation. The production of biohydrogen from various substrates are summarized in this article along with the integrated mode of dark fermentation and photo‐fermentation. Hydrogen generation through biological water‐gas shift reaction is also highlighted.
Dark fermentation and microbial fuel cells (MFCs) are two emerging technologies for biological conversion of the chemical energy of organic compounds into hydrogen (H2) and electricity, respectively. Due to kinetic and thermodynamic advantages, high temperature can be the key for increasing both dark fermentative H2 production and electricity production in MFCs. Therefore, this thesis focuses on delineating how temperature influences biological production of H2 and electricity from organic carbon-containing wastewaters. Two heat-treated inocula (fresh and digested activated sludge) were compared, for H2 production from xylose at 37, 55 and 70 °C. At both 37 and 55 °C, a higher H2 yield was achieved by the fresh than digested activated sludge, whereas a very low H2 yield was obtained by both inocula at 70 °C. Then, four different inoculum pretreatments (acidic, alkaline, heat and freezing shocks) were evaluated for creating an efficient mesophilic (37 °C) or thermophilic (55 °C) H2 producing community. Acidic and alkaline shocks selected known H2 producing microorganisms belonging to Clostridiaceae at the expenses of lactate producing bacteria, resulting in the highest H2 yield at 37 and 55 °C, respectively. Although a heat shock resulted in a low H2 yield in a single batch, H2 production by the heat-treated fresh activated sludge was shown to increase in the experiment with four consecutive batch cycles.Heat-treated fresh activated sludge was selected as inoculum for continuous H2 production from a xylose-containing synthetic wastewater in a mesophilic (37 °C) and a thermophilic (55-70 °C, increased stepwise) fluidized bed reactor (FBR). A higher H2 yield was obtained in the thermophilic than in the mesophilic FBR. Furthermore, H2 production at 70 °C, which failed in the earlier batch study, was successful in the FBR, with a stable yield of 1.2 mol H2 mol-1 xyloseadded. Operation temperature of 70 °C was also found optimal for H2 production from thermomechanical pulping (TMP) wastewater in a temperature gradient incubator assay.A RNA approach was used to study the structure and role of the anode-attached, membrane-attached and planktonic microbial communities in a mesophilic (37 °C) and a thermophilic (55 °C) two-chamber, xylose-fed MFC. An anode attached community dominated by Geobacteraceae sustained electricity production at 37 °C, whereas the establishment of methanogenic and H2 oxidizing microorganisms resulted in a low electricity production at 55 °C. However, the development of a thermophilic exoelectrogenic community can be promoted by applying a start-up strategy which includes imposing a negative potential to the anode and chemical inhibition of methanogens. A mesophilic exoelectrogenic community was also shown to produce electricity from TMP wastewater in an upflow MFC operated at 37 °C. In conclusion, a higher and more stable H2 yield can be achieved in thermophilic rather than mesophilic dark fermentation. Dark fermentation at 70 °C is particularly suitable for treatment of TMP wastewater as it is released at high temperature (50-80 °C) and could be treated on site. TMP wastewater can be also used as substrate for electricity production in mesophilic MFCs. Electricity production in thermophilic MFCs is feasible, but enrichment of thermophilic exoelectrogenic microorganisms may require a long start-up period
In this study, FW effluent was used as a substrate for both hydrogen and volatile fatty acids (VFAs) production by a lab scale set up of the semi-continuously running reactor system with a mesophilic fermentation to examine the influence of pH and pretreatment. Repeated measurement analysis showed that the factors (pH and Pretreatment) significantly influenced H2, VFAs concentration, VFA/soluble chemical oxygen demand (SCOD), and H2/SCOD traits (P < 0.0001). Duncan comparisons showed that both concentration and yield of H2 were the highest in the chemical treatment (CT) at pH 7, which were (280.82 ± 5.72) ml/L, and (4.44 ± 0.10) ml/g SCOD, respectively. While concentration and yield of the VFAs were the highest in the chemical treatment (CT) at pH 6, which were (55.44 ± 2.39) g/L, and (926.21 ± 42.27) mg/g SCOD, respectively. The butyrate and acetate for the optimal blend (pH 6, CT pretreatment) counted for 62.43% of the total VFAs.
Six moderately acidophilic, thermophilic bacterial strains with similar properties were isolated from geothermally heated water and sediment samples collected in New Zealand. These Gram stain-negative but Gram type-positive, rod-shaped bacteria formed oval terminal endospores. The cells were peritrichously flagellated and exhibited tumbling motility. At 60°C the pH range for growth was 3.8 to 6.8, and the optimum pH was 5.2 when the organisms were grown with xylose. At pH 5.2 the temperature range for growth was 35 to 66°C, and the optimum temperature was 60 to 63°C. The fermentation products from flucose or xylose were ethanol, acetate, lactate, COâ, and Hâ. The DNA G+C content was 34.5 to 35 mol%. On the basis of properties such as formation of elemental sulfur from thiosulfate, growth at acidic pH values at elevated temperatures, and the results of a 16S rRNA sequence comparison performed with previously validly published species belonging to the genus Thermoanaerobacterium, we propose that strain JW/SL-NZ613{sup T} (T = type strain) and five similar strains isolated from samples collected in New Zealand represent a new species, Thermoanaerobacterium aotearoense. Strain JW/SL-NZ613{sup T} (= DSM 10170) is the type strain of this species.
Two thermophilic, anaerobic, xylan-degrading bacteria, strains B6A-RI(T) (T = type strain) and LX-11(T), were isolated from Frying Pan Springs in Yellowstone National Park. These organisms grew chemoorganotrophically by utilizing xylan and starch but not cellulose, as well as a number of di- and monosaccharides, including glucose and xylose. Both organisms had the same optimum temperature and pH for growth (60°C and pH 6.0). The fermentation products included acetate, ethanol, lactate, CO2, and H2. Both organisms were rod shaped and deposited sulfur on their cells. The major difference between the two isolates was in spore formation; strain LX-11(T) sporulated, whereas strain B6A-RI(T) did not. Strains LX-11(T) and B6A-RI(T) were compared with other thermophilic, anaerobic, xylan-degrading bacteria by performing DNA-DNA hybridizations and total protein analyses in order to determine the relationships of these organisms. Three different groups were identified, and new taxonomic assignments are proposed. Clostridium thermocellum LQRI was least closely related to the other seven strains studied and is placed in group I, retaining its original taxonomic assignment. Clostridium thermosulfurogenes 4B(T) and new isolates B6A-RI(T) and LX-11(T) are closely related and fall into group II, for which the new genus Thermoanaerobacterium is proposed. Isolate LX-11(T) is designated Thermoanaerobacterium xylanolyticum sp. nov., and isolate B6A-RI(T) is designated Thermoanaerobacterium saccharolyticum sp. nov. Thermoanaerobacterium thermosulfurigenes 4B(T) (originally C. thermosulfurogenes) is the type strain of the type species of the genus. Group III strains are placed in the genus Thermoanaerobacter; this group includes Thermoanaerobacter ethanolicus JW200(T), Clostridium thermohydrosulfuricum 39E and E100-69(T), and Thermoanaerobium brockii HTD4(T). Thermoanaerobium brockii HTD4(T) is renamed Thermoanaerobacter brockii comb. nov., and C. thermohydrosulfuricum E100-69(T) is renamed Thermoanaerobacter thermohydrosulfuricus comb. nov. C. thermohydrosulfuricum 39E is nearly identical to Thermoanaerobacter ethanolicus JW200(T), and these organisms are considered members of the same species. Therefore, C. thermohydrosulfuricum 39E is renamed Thermoanaerobacter ethanolicus 39E; strain JW200 is the type strain of Thermoanaerobacter ethanolicus.
Cellulose in wastewater was converted into H2 by a mixed culture in batch experiments at 55 °C with various wastewaters pH (5.5-8.5) and cellulose concentrations (10-40 g 1-1). At the optimal pH of 6.5, the maximum H2 yield was 102 ml g-1 cellulose and the maximum production rate was 287 ml d-1 for each gram of volatile suspended solids (VSS). Analysis of 16S rDNA sequences showed that the cellulose-degrading mixed culture was composed of microbes closely affiliated to genus Thermoanaerobacterium.
This paper reviews information from continuous laboratory studies of fermentative hydrogen production useful when considering practical applications of the technology. Data from reactors operating with pure cultures and mixed microflora enriched from natural sources are considered. Inocula have been derived from heat-treated anaerobically digested sludge, activated sludge, aerobic compost and soil, and non-heat-treated aerobically composted activated sludge. Most studies are on soluble defined substrates, and there are few reports of continuous operation on complex substrates with mixed microflora to produce H2. Methanogenesis which consumes H2 may be prevented by operation at short hydraulic retention times (around 8– on simple substrates) and/or pH below 6. Although the reactor technology for anaerobic digestion and biohydrogen production from complex substrates may be similar, there are important microbiological differences, including the need to manage spore germination and oxygen toxicity on start-up and control sporulation in adverse circumstances during reactor operation.
Thermoanaerobacterium-rich sludge acclimated with palm oil mill effluent (POME) in an anaerobic sequencing batch reactor operating at was used as a seed in batch experiments to investigate the effects of C/N (carbon/nitrogen) ratio, C/P (carbon/phosphate) ratio and iron concentration in POME on fermentative hydrogen production. A central composite design was performed with the aim of optimizing the hydrogen yield together with POME degradation using response surface methodology (RSM). The RSM results indicated that the presence of 257 mg Fe2+/l, a C/N ratio of 74 and a C/P ratio of 559 were optimal for simultaneous hydrogen production and COD (chemical oxygen demand) removal. C/N ratio, C/P ratio and iron concentration all had an individual effect on hydrogen production and COD removal, while iron concentration and C/N ratio had the greatest interactive effect on hydrogen production (P<0.05) while C/N and C/P ratio gave more profound interactive effect on COD removal (P<0.05). The predicted maximum simultaneous hydrogen production and COD removal were 6.5 l H2/l-POME and 58%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with a hydrogen production and COD removal efficiency of H2/l-POME and 55±1.5%, respectively. The total carbohydrate conversion was 92±2.7%. The hydrogen production rate reached and increased by 60% as compared with the use of raw POME. Thermoanaerobacterium spp. were found to be dominant and present at a higher population density under optimized conditions than in raw POME fermentation. Optimization of the culture cultivation conditions in POME resulted in a simultaneous increase in biohydrogen production and COD reduction.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.
The 16S rRNA gene sequences of 34 named and unnamed clostridial strains were determined by PCR direct sequencing and were compared with more than 80 previously determined clostridial sequences and the previously published sequences of representative species of other low- G + C-content gram-positive genera, thereby providing an almost complete picture of the genealogical interrelationships of the clostridia. The results of our phylogenetic analysis corroborate and extend previous findings in showing that the genus Clostridium is extremely heterogeneous, with many species phylogenetically intermixed with other spore-forming and non-spore-forming genera. The genus Clostridium is clearly in need of major revision, and the rRNA structures defined in this and previous studies may provide a sound basis for future taxonomic restructuring. The problems and different possibilities for restructuring are discussed in light of the phenotypic and phylogenetic data, and a possible hierarchical structure for the clostridia and their close relatives is presented. On the basis of phenotypic criteria and the results of phylogenetic analyses the following five new genera and 11 new combinations are proposed: Caloramator gen. nov., with Caloramator fervidus comb. nov.; Filifactor gen. nov., with Filifactor villosus comb. nov.; Moorella gen. nov., with Moorella thermoacetica comb. nov. and Moorella thermoautotrophica comb. nov.; Oxobacter gen. nov., with Oxobacter pfennigii comb. nov.; Oxalophagus gen. nov., with Oxalophagus oxalicus comb. nov.; Eubacterium barkeri comb. nov.; Paenibacillus durum comb. nov.; Thermoanaerobacter kivui comb. nov.; Thermoanaerobacter thermocopriae comb. nov.; and Thermoanerobacterium thermosaccharolyticum comb. nov.
Two anaerobic, thermophilic, Gram-positive, non-spore forming bacteria with an array of polysaccharide-degrading enzymes were isolated from the leachate of a waste pile from a canning factory in Hoopeston, East Central Illinois, USA. The results of 16S rDNA sequence homology indicated that their closest relatives belong to the saccharolytic, thermophilic and anaerobic genera of Thermoanaerobacterium and Thermoanaerobacter. Although, the evolutionary distances between these bacteria and their closest relatives are greater than 11%, there is no defining phenotypic characteristic for the creation of a new genus. It is proposed that these bacteria should be placed in the genus Thermoanaerobacterium, which requires emendment of the genus description with regard to the reduction of thiosulfate to sulfur, because neither isolate is capable of this reduction. Thermoanaerobacterium polysaccharolyticum reduces thiosulfate to sulfide, whereas Thermoanaerobacterium zeae is unable to reduce thiosulfate. The cells of both isolates are rod-shaped and exist as single cells or sometimes in pairs. Cells are motile by means of flagella. Growth occurs between 45 and 72 degrees C, with optimum temperature of 65-68 degrees C at pH 6.8. The pH range for growth is from 4 to 8 at a temperature of 65 degrees C. Both organisms ferment glucose, arabinose, maltose, mannose, rhamnose, sucrose, trehalose, xylose, cellobiose, raffinose, melibiose and melezitose. The major end products of fermentation with glucose are ethanol and CO2, with lesser amounts of acetate, formate, lactate and hydrogen. The DNA G+C contents of Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov. are 46 and 42 mol%, respectively. The type strains are KMTHCJT (= ATCC BAA-17T = DSM 13641T) and mel2T (= ATCC BAA-16T = DSM 13642T), respectively.
A new, extremely thermophilic bacterium, designated strain MB4T, was isolated from a Chinese hot spring. The new isolate was an obligately anaerobic, rod-shaped, gram-negative, saccharolytic bacterium. Spore formation was not observed. Growth occurred at temperatures between 50 and 80 degrees C, with an optimum of around 75 degrees C; at pH values between 5.5 and 9.0, with an optimum of 7.0-7.5; and at salinities between 0 and 2.5% NaCl, with an optimum of around 0.2% NaCl. The organism utilized glucose, galactose, maltose, cellobiose, mannose, fructose, lactose, mannitol and starch. Acetate was the main end product from glucose fermentation. Thiosulfate and sulfur were reduced to hydrogen sulfide. Sulfate, sulfite and nitrate were not reduced. Growth was inhibited by hydrogen. The G+C content of the DNA was 33 mol%. Phylogenetic analyses based on the 16S rDNA sequence indicated that the isolate was a new member of the genus Thermoanaerobacter and formed a monophyletic unit within the Thermoanaerobacter cluster. Based on its phenotypic and phylogenetic characteristics, the isolate was proposed as a new species, Thermoanaerobacter tengcongensis. The type strain is MB4T (= Chinese Collection of Microorganisms AS 1.2430T = JCM 11007T).
A hydrogen-producing sludge degraded 99% of glucose at 36 degrees C and pH 5.5, producing a methane-free biogas (comprising 64% hydrogen) and an effluent comprising mostly butyrate, acetate, and ethanol. The yield was 0.26 l H2 g(-1) glucose and the production rate per gram of volatile suspended solids was 4.6 1 H2 day(-1). A 16S rDNA library was constructed from the sludge for microbial species determination. A total of 96 clones were selected for plasmids recovery, screened by denaturing gradient gel electrophoresis, and sequenced for rDNA. Based on the phylogenetic analysis of the rDNA sequences, 64.6% of all the clones were affiliated with three Clostridium species (Clostridiaceae), 18.8% with Enterobacteriaceae, and 3.1% with Streptococcus bovis (Streptococcaceae). The remaining 13.5% belonged to eight operational taxonomic units, the affiliations of which were not identified.
Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively. Both strains, together with Thermoanaerobacter yonseiensis KB-1(T) (=DSM 13777(T)), Thermoanaerobacter tengcongensis MB4(T) (=DSM 15242(T)) and Carboxydibrachium pacificum JM(T) (=DSM 12653(T)), possessed genomic (DNA-DNA hybridization studies) and phylogenetic similarities with Thermoanaerobacter subterraneus SEBR 7858(T) (=DSM 13054(T)), which was isolated recently from an oilfield reservoir in south-west France. Marked phenotypic differences exist between the three oilfield isolates (T. subterraneus, strain OCA1 and strain SL9): they include temperature range for growth and substrates used. Differences were also observed in the DNA G+C contents of all organisms. Similarly to T. subterraneus, strains SL9 and OCA1, and also T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum, produced acetate and L-alanine as major end products of glucose metabolism [0.8-1.0 mol L-alanine produced (mol glucose consumed)(-1)] and reduced thiosulfate, but not sulfate, to sulfide. Because of these significant metabolic and phylogenetic differences between the oilfield isolates (T. subterraneus, strain OCA1 and strain SL9), T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum and other Thermoanaerobacter species, it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov., with the creation of four novel subspecies, Caldanaerobacter subterraneus subsp. subterraneus subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. yonseiensis subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. tengcongensis subsp. nov., comb. nov. and Caldanaerobacter subterraneus subsp. pacificus subsp. nov., comb. nov.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
Sewage sludge was acclimated to establish H-2-producing enrichment cultures for converting sucrose (20 g COD/l) into H-2 in an up-flow anaerobic sludge blanket (UASB) reactor. The operating hydraulic retention times (HRTs) were 24-4 h. The experimental results indicated that this UASB system could be used for hydrogen production. The hydrogen productivity was HRT dependent and nearly constant at the HRT of 8-20 h. However, it drastically decreased at an HRT of 4 or 24 h. The hydrogen production rate (HPR) and specific HPR peaked at the HRT of 8 h and drastically decreased at all other HRTs. At an HRT of 8 h, the average granular diameter peaked at 0.43 mm and each gram of biomass produced 53.5 mmol H-2/day with a hydrogen gas content of 42.4% (v/v). Butyrate and acetate were the main fermentation volatile fatty acids. The anaerobic granule sludge kinetic constants were endogenous decay coefficient (K-d) 0.1 day(-1) and yield coefficient (Y-g) 0.1 g VSS/g COD. The mean cell retention time was 22.2 h and the excess sludge discharge rate was 3.24 l/day.
Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively. Both strains, together with Thermoanaerobacter yonseiensis KB-1 T ( = DSM 13777 T ), Thermoanaerobacter tengcongensis MB4 T (= DSM 15242 T ) and Carboxydibrachium pacificum JM T ( = DSM 12653 T ), possessed genomic (DNA-DNA hybridization studies) and phylogenetic similarities with Thermoanaerobacter subterraneus SEBR 7858 T (= DSM 13054 T ), which was isolated recently from an oilfield reservoir in south-west France. Marked phenotypic differences exist between the three oilfield isolates (T. subterraneus, strain OCA1 and strain SL9): they include temperature range for growth and substrates used. Differences were also observed in the DNA G+C contents of all organisms. Similarly to T. subterraneus, strains SL9 and OCA1, and also T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum, produced acetate and L-alanine as major end products of glucose metabolism [0.8-1.0 mol L-alanine produced (mol glucose consumed) -1 ] and reduced thiosulfate, but not sulfate, to sulfide. Because of these significant metabolic and phylogenetic differences between the oilfield isolates (T. subterraneus, strain OCA1 and strain SL9), T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum and other Thermoanaerobacter species, it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov., with the creation of four novel subspecies, Caldanaerobacter subterraneus subsp. subterraneus subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. yonseiensis subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. tengcongensis subsp. nov., comb. nov. and Caldanaerobacter subterraneus subsp. pacificus subsp. nov., comb. nov.
Based on comparative analyses of 16S and 23S ribosomal RNA sequences we have located sites specific for the alpha-, beta-, and gamma-subclasses of Proteobacteria. Short oligodeoxynucleotides complementary to these signature regions were evaluated as potential nucleic acid probes for the differentiation of the major subclasses of Proteobacteria. Hybridization conditions were optimized by the addition of formamide to the hybridization buffer and high stringency post-hybridization washing. Single-mismatch discrimination of probes was further improved by blocking nontarget probe binding sites with competitor oligonucleotides. Nonisotopic dot-blot hybridization to reference strains demonstrated the expected probe specificities, whole cell hybridization with fluorescent probe derivatives allowed the classification of individual microbial cells. The probes will be useful for determinative studies and for the in situ monitoring of population distribution and dynamics in microbial communities.
Hyper-thermophilic hydrogen production without methane was demonstrated for the first time in granular up-flow anaerobic sludge blanket (UASB) system fed with glucose using mixed cultures. The maximum hydrogen yield in this study was 2.47 +/- 0.15 mol H2/mol glucose. This high yield has never been previously reported in mixed culture systems and it was likely due to more favorable thermodynamic conditions at hyper-thermophilic temperatures. Different start-up strategies (bromoethanosulfonate (BES) addition and flow recycle) were evaluated. BES addition during start-up prevented the establishment of methanogenic cultures in granules. Flow recycle was important to achieve higher hydrogen yield through enriching better hydrogen-producing organisms and reduced the start-up period as well. This study indicated UASB system was a promising system for hydrogen production.
In this article the role of hydrogen as a process monitoring tool in methanogenic systems was studied by considering the influence of several key system parameters. Hydrogen production was found to be influenced mainly by the inocula's source pH, and varied only slightly with external pH and HCO3− levels. When an inoculum adapted to above neutral conditions (pH>7) was shocked, reducing equivalents were selectively channelled through formate, while high hydrogen production was noticed with acidically (pH
As a new clean energy source, the demands for and use of hydrogen fuel are rapidly increasing. Therefore, biohydrogen production technology is being developed to reduce operation costs in many countries. Improvement of biohydrogen production capacity and cost reduction are key factors to bring about industrial implementation. One of the most effective production methods is microbiological: the use of bacteria with high hydrogen-production capacity and performance. The anaerobic process of biohydrogen production was developed in the 1990s. The isolation and identification of highly efficient biohydrogen producing anaerobic bacteria is an important foundation for the fermentative production of hydrogen by anaerobic digestion of organic wastewater.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Hydrogen can be harvested from the microbial fermentation of organic substrates when methanogenesis is suppressed in an anaerobic digestion system. In this study three methods, heat-, acid- and alkaline-treatment, were used to suppress methanogenesis in mixed cultures and to enrich H2-producing inoculum. Highest H2 yield of 2.00 mol-H2/mol-glucose was achieved with the heat-treated sludge, while lowest yield of 0.48 mol-H2/mol-glucose was obtained with the alkaline-treated sludge. A butyrate-type fermentation was found for both heat- and alkaline-treated sludge, while a mixed-type fermentation occurred for the acid-treated sludge. A model was established to describe the kinetics of H2 production process and the yield coefficients of various products were estimated for the three cases with this model. The relationships among NADH/NAD+, oxidation–reduction potential and the H2 partial pressure were established and the evolvement of NADH/NAD+ and oxidation–reduction potential in the fermentative process for the three cases was also evaluated. The comparative experimental results show that the heat-treatment method was better than the two others for enriching H2-producing inoculums from mixed anaerobic cultures.
A base-enriched anaerobic mixed microflora was used for hydrogen fermentation from sucrose in a laboratory scale model completely stirred tank bioreactor operating at 35 °C. The purpose of the study was to determine the microflora hydrogenic activity and its effects from the change of hydraulic retention time (HRT, 2–12 h). The experimental results indicate that base-enriched mixed microflora could be used as the seed for efficient hydrogen fermentation. The fermenter could operate stably for 250 days at a HRT of 12 h. Hydrogen gas content, hydrogen productivity and hydrogen production rate were HRT-dependent and their values ranged 38.7–45.9%, 0.9–3.5 mol H2/mol sucrose and 263–408 mmol H2/L day, respectively, with a HRT of 4 h having peak hydrogen production. The biomass activity was also HRT-dependent with each gram of biomass producing 65–145 mmol H2/day. The DGGE analysis shows that the microbial species shifted during the HRT-reduction operation but Clostridium ramosum was dominant. Those hydrogen productivity values were comparable to the hydrogen production using pure cultures, other mixed microflora or other reactor systems. The major liquid fermentation products were ethanol, acetic, propionic and butyric acids; their concentrations were also HRT-dependent. Strategies based on these results for optimal hydrogen production were proposed.
We evaluated the influence of the operation temperature (mesophilic vs. thermophilic regime) of semicontinuous, acidogenic solid substrate anaerobic digestion (A-SSAD) of the organic fraction of municipal solid waste (OFMSW) at lab scale. The H2 percentage was higher in the thermophilic regime than in the mesophilic operation (58% and 42%, respectively). The H2 yield of thermophilic A-SSAD was significantly higher than in our mesophilic reactors (360 vs. 165 NmL H2/g VSrem) and other studies reported in the literature (range of 62–180 mL/g VS). Mesophilic A-SSAD showed a yield of 37% of the maximum yield based on 4 mol H2/mol hexose, while thermophilic A-SSAD exhibited a yield of 80% of the maximum yield. This result is similar to works with pure cultures of extremophile microorganisms where H2 yields of 83% of the maximum were reported. We found higher concentrations of acetic acid in the digestates of thermophilic A-SSAD, while butyrate was in higher proportion in mesophilic A-SSAD spent solids. The moderate-to-high yields obtained with the semicontinuous process used in this work are in disagreement with previous reports claiming that batch and semicontinuous processes are less efficient than continuous ones.
Sewage sludge was acclimated to establish H2-producing enrichment cultures for converting sucrose into H2 in an up-flow anaerobic sludge blanket (UASB) reactor. The operating hydraulic retention times (HRTs) were 24–. The experimental results indicated that this UASB system could be used for hydrogen production. The hydrogen productivity was HRT dependent and nearly constant at the HRT of 8–. However, it drastically decreased at an HRT of 4 or . The hydrogen production rate (HPR) and specific HPR peaked at the HRT of and drastically decreased at all other HRTs. At an HRT of , the average granular diameter peaked at and each gram of biomass produced with a hydrogen gas content of 42.4% (v/v). Butyrate and acetate were the main fermentation volatile fatty acids. The anaerobic granule sludge kinetic constants were endogenous decay coefficient and yield coefficient . The mean cell retention time was and the excess sludge discharge rate was .
Chloroform (CHCl3), 2-bromoethanesulfonate (BES) and fluoroacetate have frequently been used as methanogenic inhibitors in rice field soil and in other environments, but their effects on other microbial processes have not received sufficient attention. Therefore, we comparatively determined the effects of CHCl3, BES and fluoroacetate on different microbial processes in rice field soil slurry upon incubation under anoxic conditions: on the reduction of the electron acceptors nitrate, ferric iron, sulfate; on the production of CH4 and CO2; on the temporal change of the electron donors H2, acetate and propionate; and on the turnover of [2-]acetate during the early reduction phase (day 7), and during the later methanogenic phase (day 30). The results demonstrate: (1) fluoroacetate inhibited acetate consumption by all microorganisms, (2) BES generally inhibited CH4 production, and (3) CHCl3 not only inhibited methanogenesis, but partially also acetate-dependent sulfate reduction, and perhaps H2-dependent homoacetogenesis. The specificity of the different inhibitors resulted in characteristic patterns of the temporal change of electron donors and acceptors and of CH4. The pattern of propionate change was consistent with production by fermenting bacteria and consumption by sulfate reducers either using sulfate or methanogens as electron acceptor. Sulfate reducers were also found to be important for acetate consumption during the early phase of soil anoxia. Later on, however, methanogenic acetate consumption was much more pronounced. The application of inhibitors with different specificity was helpful for elucidating the flow of carbon and electrons during degradation of organic matter in anoxic rice field soil.
Growth and hydrogen production by two extreme thermophiles during sugar fermentation was investigated. In cultures of Caldicellulosiruptor saccharolyticus grown on sucrose and Thermotoga elfii grown on glucose stoichiometries of of hydrogen and of acetate per mol C6-sugar unit were obtained. The hydrogen level was about 83% of the theoretical maximum. C. saccharolyticus and T. elfii reached maximum cell densities of 1.1×109 and 0.8×109 cells/ml, respectively, while their maximum hydrogen production rates were 11.7 and dry weight/h, respectively. For growth of C. saccharolyticus on sucrose, a biomass yield of sucrose and a YATP of 11.3–14.1 were calculated. Replacement of yeast extract by casamino acids, plus proline and vitamins in the medium of C. saccharolyticus resulted in similar yields of hydrogen production on sucrose, but diminished the rate by about 30%. Both yeast extract and tryptone were required by T. elfii, and appeared to function as sources of carbon, nitrogen and energy. In the absence of tryptone, T. elfii converted 26% of the glucose to another by-product, resulting in a lower yield of hydrogen. Growth of T. elfii ceased prior to glucose depletion, but the culture continued to ferment glucose to hydrogen and acetate until all glucose was consumed.
Six digested sludges, pre-treated by different methods (heat-shock, aeration, acid and base treatments, 2-bromoethanesulfonic acid (BESA) inhibition and iodopropane inhibition) as well as an untreated sample were compared for their suitability in the preparation of hydrogen producing seeds by cultivations in a sucrose medium. The heat-shock and acid treatment methods completely repressed methanogenic activity; however, they also partially repressed hydrogen production. The base treatment option did not completely repress methanogenic activity and also significantly impacted hydrogen production. The aeration method was unsuccessful at completely repressing methanogenic activity; however, it did not significantly affect the hydrogen production activity. The BESA and iodopropane pre-treatment methods specifically inhibited the methanogens, and there were no significant effects found on hydrogen production. Similar to the aeration pre-treated digested sludge sample, the untreated sludge showed high hydrogen production activity and a small amount of methanogenic activity (lower than the activity detected in the base treatment sample). In the subsequent second-step batch cultivations with the same sucrose medium and the diluted media, methanogenic activity was not detected in any of the test bottles. The microbial seed prepared from base treatment exhibited the highest hydrogen production activity, whereas those prepared from acid treatment did not exhibit any activity. Again, the microbial seed prepared from untreated sludge also exhibited relatively high hydrogen producing activity. A lower pH was detected at the end of the cultivation in all the test bottles. Interestingly, the variations in pH in the different tests bottles indicate that pH is an important parameter in the control of methanogenic activity.
The hydrogen production yield from glucose by an isolate was investigated and compared to that by microflora. The isolate, Thermoanaerobacterium thermosaccharolyticum KU001, from the microflora demonstrated approximately 2.4 mol/mol-glucose of hydrogen production with acetate/butyrate formation in an artificial medium. The fermentation pattern was similar to that observed for the hydrogen fermentation of wastewater by the microflora. A PCR-DGGE analysis of the bacterial 16S rDNA detected T. thermosaccharolyticum in the microflora with strong intensity of the characteristic 16S rDNA band, although the microflora was enriched from an artificial medium. These results imply that T. thermosaccharolyticum could be a predominant species of the microflora that is involved in hydrogen-producing acetate/butyrate fermentation. The nitrogen source in the medium affected the carbohydrate metabolism of KU001, and caused a change in hydrogen yield.
This paper investigated hydrogen production from a model lignocellulosic waste in inhibited solid substrate anaerobic digesters. Acetylene at 1% in the headspace was as effective as bromoethanesulfonate in inhibiting methanogenic activity in batch anaerobic composters containing 25% () total organic solids inoculated with an undefined cellulotytic consortium derived from anaerobic digesters. Acetylene also had no effect on the rate and amount of hydrogen produced from a pure culture of Clostridium thermocellum grown under the same conditions.
Hydrogen systems can provide viable, sustainable options for meeting the world's energy requirements. Hydrogen is relevant to all of the energy sectors—transportation, buildings, utilities and industry. It can provide storage options for baseload (geothermal), seasonal (hydroelectric) and intermittent (PV and wind) renewable resources, and when combined with emerging decarbonization technologies, can reduce the climate impacts of continued fossil fuel utilization. However, hydrogen energy systems still face a number of technical and economical barriers that must first be overcome for hydrogen to become a competitive energy carrier. Advances must be made in hydrogen production, storage, transport and utilization technologies and in the integration of these components into complete energy systems. To expedite the advancement of hydrogen technologies and realize a hydrogen future, nations have come together under the auspices of the International Energy Agency's (IEA) Hydrogen Program to collaborate and address the important barriers that impede hydrogen's worldwide acceptance. Through well-structured, collaborative projects, experts from around the world address many of the technical challenges and long-term research needs that face the hydrogen community. These collaborations have already led to significant advances in renewable hydrogen production and solid storage materials and to the development of tools to evaluate and optimize integrated hydrogen energy systems.
Palm oil mill effluent (POME) sludge, sludge compost from Malaysia and CREST compost from Philippines were collected for the study. The capability of these microflora to produce hydrogen was examined with artificial wastewater containing 1% glucose, 0.2% yeast extract and 0.018% magnesium chloride hexahydrate under anaerobic fermentation in a batch culture. The microflora in POME sludge, sludge compost and CREST compost were found to produce significant amounts of hydrogen. The maximum production yield of hydrogen per decomposed glucose was -glucose at a conversion rate of at 50°C obtained by sludge compost. All fermentations were carried out without pH control. It was also found that the addition of nitrogen source in the medium caused a change in hydrogen produced. There was no methane gas in the evolved gas.
The influence of the chemical nature of high-solid organic wastes (HSOW) on their biohydrogen generation was investigated using simulated high-solid bioreactors under mesophilic conditions. The bioreactors were filled with 10% total solid of rice, potato, fat meat, chicken skin, egg, and lean meat. Experimental results indicate that hydrogen-producing potential of carbohydrate-rich HSOW (rice and potato) was approximately 20 times larger than that of fat-rich HSOW (fat meat and chicken skin) and of protein-rich HSOW (egg and lean meat). According to development trends of pH and hydrogen, pH around 6.0 might be threshold for heat-shock digested sludge; that is Clostridium-rich sludge, converting fat- and protein-rich HSOW to hydrogen; but pH threshold for Clostridium-rich sludge consuming carbohydrates-rich HSOW occurred at around 5.0. In bulk solution, volatile fatty acids (VFA) and alcohols occurred concurrently and the trends of carbohydrate-rich HSOW were similar to those of protein-rich HSOW. Considering developments of carbohydrates and VFAs together with that of hydrogen one infers that lipids would be hydrolyzed to carbohydrates and the carbon flow would proceed through acetate/H2+CO2 cleavage. Indications from cluster analysis of pH development trends are that a cometabolism would be obtained in wastes rich in carbohydrate and protein.
In this research, batch and continuous-flow reactors were used to study biological hydrogen production by anaerobic mixed communities subjected to two types of heat treatment--initial and repeated heat treatment. The bioreactors were inoculated with anaerobically digested municipal sludge, and sucrose was used as substrate. In the batch experiments, applying heat treatment at 100⁰C for 15 min. to the seed inoculum (initial heat treatment) was found necessary to sustain hydrogen production, and continuously applying heat treatment at 100⁰C for 15 min. to the sludge (repeated heat treatment) could promote higher hydrogen production. In the continuous-flow experiments, besides applying initial heat treatment at 100⁰C for 15 min. to the inoculum, repeated heat treatment at 90⁰C for 20 min. was applied to the return sludge using external heated chamber. Results indicated that both initial and repeated heat treatments promoted hydrogen production by eliminating non-spore-forming, hydrogen-consuming microorganisms and by selecting for hydrogen-producing, spore-forming bacteria. It was also observed that the hydrogen production efficiency was superior at a higher feed concentration (0.13 L-H₂/g COD at 20 g COD/L) in comparison to a lower feed concentration (0.07 L-H₂/g COD at 6 g COD/L). Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that Clostridium and Bacillus species were dominant populations in the bioreactors. A positive correlation was observed between the total abundance of Clostridium species and hydrogen production during part of an operational run. Typescript (photocopy). Thesis (M.S.)--Iowa State University, 2002. Includes bibliographical references.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
In this article the role of hydrogen as a process monitoring tool in methanogenic systems was studied by considering the influence of several key system parameters. Hydrogen production was found to be influenced mainly by the inocula's source pH, and varied only slightly with external pH and HCO3- levels. When an inoculum adapted to above neutral conditions (pH > 7) was shocked, reducing equivalents were selectively channelled through formate, while high hydrogen production was noticed with acidically (pH < 6.5) adapted inocula. The results also revealed that the production of hydrogen or formate during shock loads was not strongly associated with microbial morphology (granules or flocs) as high electron fluxes were possible through either during acidogenesis. Shock load experiments in continuous reactors revealed that neither hydrogen nor formate accumulated to any significant degree, nevertheless digester recovery took a long time due to the slow kinetics of volatile fatty acid degradation. Selective formate production under neutral pH environments, coupled with high hydrogenotrophic activity, was found to be responsible for the dampened hydrogen response during the early phases of gradually shocked systems (step change). Based on these results it appears that the role of hydrogen as a process monitoring tool has been overemphasised in the literature.
The conversion of organics in wastewaters into hydrogen gas could serve the dual role of renewable energy production and waste reduction. The chemical energy in a sucrose rich synthetic wastewater was recovered as hydrogen gas in this study. Using fractional factorial design batch experiments, the effect of varying pH (4.5-7.5) and substrate concentration (1.5-44.8 g COD/L) and their interaction on hydrogen gas production were tested. Mixed bacterial cultures obtained from a compost pile, a potato field, and a soybean field were heated to inhibit hydrogen-consuming methanogens and to enrich sporeforming, hydrogen-producing acidogens. It was determined that the highest rate (74.7 mL H2/(L*h)) of hydrogen production occurred at a pH of 5.5 and a substrate concentration of 7.5 g COD/Lwith a conversion efficiency of 38.9 mL H2/(g COD/L). The highest conversion efficiency was 46.6 mL H2/(g COD/L).
The activated sludge comprises a complex microbiological community. The structure (what types of microorganisms are present) and function (what can the organisms do and at what rates) of this community are determined by external physico-chemical features and by the influent to the sewage treatment plant. The external features we can manipulate but rarely the influent. Conventional control and operational strategies optimise activated sludge processes more as a chemical system than as a biological one. While optimising the process in a short time period, these strategies may deteriorate the long-term performance of the process due to their potentially adverse impact on the microbial properties. Through briefly reviewing the evidence available in the literature that plant design and operation affect both the structure and function of the microbial community in activated sludge, we propose to add sludge population optimisation as a new dimension to the control of biological wastewater treatment systems. We stress that optimising the microbial community structure and property should be an explicit aim for the design and operation of a treatment plant. The major limitations to sludge population optimisation revolve around inadequate microbiological data, specifically community structure, function and kinetic data. However, molecular microbiological methods that strive to provide that data are being developed rapidly. The combination of these methods with the conventional approaches for kinetic study is briefly discussed. The most pressing research questions pertaining to sludge population optimisation are outlined.
This study offers a novel and quick enrichment technology that can be used as a preliminary method to obtain a hydrogen-producing species from the biological sludge produced by wastewater treatment. The influences of acid-base enrichment (by sludge pH adjustment) on the anaerobic hydrogen-producing micro-organisms were investigated using serum bottle assays. The enrichment pH values were controlled at 3, 4, 5, 7, 10, 11 and 12 with 1 N hydrochloric acid and 1 N sodium hydroxide. For each enrichment pH, the cultivation pH values were controlled at 5, 6 and 7. Based on the experimental results, hydrogen accumulation from sludge with acid or base enrichment is higher than that of the control. The hydrogen-production potential of the sludge with acid or base enrichment is 200 and 333 times enhanced, compared with the control, when the enrichment pH is 10 and 3, respectively. The enhancement is due to a shortening of the micro-organisms' lag-time which occurs at a proper cultivation-pH level.
Batch experiments were conducted to convert starch in wastewater into hydrogen at 55 degrees C at various wastewater pH (4.0-9.0) and starch concentrations (9.2-36.6 g/l). The maximum hydrogen yield of 92 ml/g of starch added (17% of the theoretical value) was found at wastewater pH 6.0, and the maximum specific hydrogen production rate of 365 ml/(g-VSS.d) was at wastewater pH 7.0. The methane-free biogas contained up to 60% of hydrogen. The mixed liquor was composed mostly of acetate (40.2-53.4%) and butyrate (26.0-40.9%). Phylogenetic analysis based on 16S rDNA sequences of the 72 clones developed from the sludge at pH 6.0 shows that 85.7% of the clones were closely affiliated with genus Thermoanaerobacterium in family Thermoanaerobacteriaceae; the remaining 14.3% were with an uncultured Saccharococcus sp. clone ETV-T2.
Batch tests were carried out to analyze influences of the alkaline pretreatment and initial pH value on biohydrogen production from sewage sludge. Experimental results of the impact of different initial pH on biohydrogen production showed that both the maximal hydrogen yield occurred and that no methane was detected in the tests of at the initial pH of 11.0. The final pH decreased at the initial pH of 7.0-12.5 but increased atthe initial pH of 3.0-6.0, probably due to the combination of solubilized protein from sludge and the formation of volatile fatty acids (VFAs) and ammonia during biohydrogen fermentation. The performance of biohydrogen production using the raw sludge and the alkaline pretreated sludge was then compared in batch fermentation tests atthe initial pH of 11.0. The hydrogen yield was increased from 9.1 mL of H2/g of dry solids (DS) of the raw sludge to 16.6 mL of H2/g of DS of the alkaline pretreated sludge. No methane and less carbon dioxide (0.8% of control) were present in the biohydrogen production from the alkaline pretreated sludge. These results clearly showed that biohydrogen production could be enhanced and maintained stable by the combination of the high initial pH and alkaline pretreatment. The mechanism of biohydrogen production from sewage sludge at high initial pH was therefore investigated because the results of this study were differentfrom previous studies of biohydrogen production. Results showed that protein was the major substrate for biohydrogen production from sewage sludge and that Eubacterium multiforme and Paenibacillus polymyxa were the dominant bacteria in biohydrogen production from alkaline pretreated sludge at an initial pH of 11.0. The combination of alkaline pretreatment and high initial pH could not only maintain a suitable pH range for the growth of dominant hydrogen-producing anaerobes but also inhibit the growth of hydrogen-consuming anaerobes. In addition, the changes in pH value, oxidation-reduction potential, VFAs and soluble COD during hydrogen fermentation were also discussed.