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Effects of heat treatment on hydrogen production potential and microbial community of thermophilic compost enrichment cultures
Abstract
Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. In this study, hydrogen producing, cellulolytic cultures were enriched from compost material at 52, 60 and 70°C. Highest cellulose degradation and highest H(2) yield were 57% and 1.4 mol-H(2) mol-hexose(-1) (2.4 mol-H(2) mol-hexose-degraded(-1)), respectively, obtained at 52°C with the heat-treated (80°C for 20 min) enrichment culture. Heat-treatments as well as the sequential enrichments decreased the diversity of microbial communities. The enrichments contained mainly bacteria from families Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen production and bacteria closely related to Clostridium cellulosi and Clostridium stercorarium were responsible for cellulose degradation.
... For example, with microbial consortia it is feasible to convert wastewater Lin et al., 2012a;Yossan et al., 2012; and food wastes (Valdez-Vazquez et al., 2005;Kim et al., 2009;Ohnishi et al., 2010;Sreela-or et al., 2011; into H 2 , a task economically restrictive with pure cultures. Also, microbial consortia act cooperatively to hydrolyze and ferment polysaccharides with different grades of complexity, from starch to lignocellulosic biomass (Akutsu et al., 2008;Lin and Hung, 2008;Nissilä et al., 2011aNissilä et al., , 2011bChen et al., 2012;Lay et al., 2012;Gadow et al., 2013;Valdez-Vazquez et al., 2015a). On the other side of the coin, H 2 -producing microbial consortia have significant drawbacks that may limit their performance and application in full-scale bioreactors. ...
... Authors have sampled different environments, such as anaerobic sludge (Koskinen et al., 2007;Davila-Vazquez et al., 2009;Baghchehsaraee et al., 2010;Yossan et al., 2012;Sivagurunathan et al., 2013), compost (Akutsu et al., 2008;Huang et al., 2010;Nissilä et al., 2011aNissilä et al., , 2011b, and wastewater treatment plants (Akutsu et al., 2009;Baghchehsaraee et al., 2010;Chaganti et al., 2012;Lay et al., 2012;Sivagurunathan et al., 2013) with the aim of selecting an active microbial community able to produce H 2 . Since in these consortia non-H 2 -producing bacteria, H 2 -producing bacteria, and H 2 -consuming bacteria coexist, the literature is full of methods and strategies for enriching the inocula with H 2 -producing bacteria (Baghchehsaraee et al., 2008;Ren et al., 2008a;Adav et al., 2009;Hafez et al., 2009;Kim et al., 2009;O-Thong et al., 2009;Ohnishi et al., 2010;Nasr et al., 2011;Nissilä et al., 2011aNissilä et al., , 2011bRossi et al., 2011;Li et al., 2012b;Ning et al., 2012;Jeong et al., 2013;Wan et al., 2013). ...
... Authors have sampled different environments, such as anaerobic sludge (Koskinen et al., 2007;Davila-Vazquez et al., 2009;Baghchehsaraee et al., 2010;Yossan et al., 2012;Sivagurunathan et al., 2013), compost (Akutsu et al., 2008;Huang et al., 2010;Nissilä et al., 2011aNissilä et al., , 2011b, and wastewater treatment plants (Akutsu et al., 2009;Baghchehsaraee et al., 2010;Chaganti et al., 2012;Lay et al., 2012;Sivagurunathan et al., 2013) with the aim of selecting an active microbial community able to produce H 2 . Since in these consortia non-H 2 -producing bacteria, H 2 -producing bacteria, and H 2 -consuming bacteria coexist, the literature is full of methods and strategies for enriching the inocula with H 2 -producing bacteria (Baghchehsaraee et al., 2008;Ren et al., 2008a;Adav et al., 2009;Hafez et al., 2009;Kim et al., 2009;O-Thong et al., 2009;Ohnishi et al., 2010;Nasr et al., 2011;Nissilä et al., 2011aNissilä et al., , 2011bRossi et al., 2011;Li et al., 2012b;Ning et al., 2012;Jeong et al., 2013;Wan et al., 2013). ...
... The polysaccharolytics identii fied in thermophilic reactors operating at 50–55°C were related to the species isolated from both anthroo pogenic (digested sludge, compost, fermented manure, and contaminated soils) and natural habitats (water, mud and sediments of geothermally heated pools and thermal springs). Clostridium species prevailed in the first case, while Petrotoga and Thermoo anaerobacterium species were found in the second case (Table 2 [18, 27, 32, 36, 38,4142434445464748495051). Polysaccharolyy tics identified in hydrogen bioreactors operating at temperatures above 65°C were related to extremely thermophilic members of the genera Caloramator, Caldanaerobacter, Thermoanaerobacter, and Therr moanaerobacterium, which have been isolated mostly from natural habitats (geothermally heated streams and thermal springs) (Table 3 [18, 36, 38, 40, 42, 45, 52, 53]). ...
... Clostridium species prevailed in the first case, while Petrotoga and Thermoo anaerobacterium species were found in the second case (Table 2 [18, 27, 32, 36, 38,4142434445464748495051). Polysaccharolyy tics identified in hydrogen bioreactors operating at temperatures above 65°C were related to extremely thermophilic members of the genera Caloramator, Caldanaerobacter, Thermoanaerobacter, and Therr moanaerobacterium, which have been isolated mostly from natural habitats (geothermally heated streams and thermal springs) (Table 3 [18, 36, 38, 40, 42, 45, 52, 53]). In reactors operating at 30–35°C, both mesophilic and moderately thermophilic polysaccharolytics occur. ...
... A thermophilic proteolytic Coprotherr mobacter platensis was isolated from a mesophilic UASB reactor treating wastewater of a baker's yeast production facility [57]. Another species Coprotherr mobacter proteolyticus, which was isolated from a therr mophilic reactor fermenting tannery waste and cattle manure [58], is most often detected by molecular techniques in various waste treatment systems [36, 45, 59]. Many proteolytic bacteria are also capable of carr bohydrate fermentation. ...
Methane production via anaerobic degradation of organic-contaminated wastewater, semiliquid, or solid municipal waste of complex composition by methanogenic microbial communities is a multistage process involving at least four groups of microorganisms. These are hydrolytic bacteria (polysaccharolytic, proteolytic, and lipolytic), fermentative bacteria, acetogenic bacteria (syntrophic, proton-reducing), and methanogenic archaea; complex trophic interactions exist between these groups. The review provides information concerning the diversity of the major microbial groups identified in the systems for wastewater and concentrated waste treatment, solid-phase anaerobic fermentation, and landfills for disposal of municipal solid waste, and also specifies the sources of isolation of the type strains. The research demonstrates that both new microorganisms and those previously isolated from natural habitats may be found in waste treatment systems. High microbial diversity in the systems for organic waste treatment provides for stable methanogenesis under fluctuating environmental conditions.
... Schlüter). thermocellum were responsible for cellulose degradation (Nissila et al., 2011;Ueno et al., 2001). From these, R. cellulosi and R. stercorarium most probably are the two main species responsible for lignocellulose degradation (Nissila et al., 2011). ...
... thermocellum were responsible for cellulose degradation (Nissila et al., 2011;Ueno et al., 2001). From these, R. cellulosi and R. stercorarium most probably are the two main species responsible for lignocellulose degradation (Nissila et al., 2011). Zhang et al. (2014) isolated new strains of R. stercorarium (CS-3-2) and R. cellulosi (CS-4-4) from decayed corn stalk. ...
... To date, R. cellulosi has not been detected as part of microbial communities of biogas reactors as analyzed in corresponding metagenome studies Schlüter et al., 2008;Wirth et al., 2012). However, recent results from studies addressing decayed cornstalk and compost microbial communities indicated that this species seems to play an important role in substrate hydrolysis in these communities (Nissila et al., 2011;Ueno et al., 2001;Zhang et al., 2014). ...
... Hydrogen gas has many advantages; it has most energy per unit mass (122 MJ/kg), and it can be used for electricity production in fuel cells with water and heat as the only endproducts [1] [2]. Dark fermentative hydrogen productions from both pure cellulosic materials, such as cellulose and xylan [3] [4], and renewable cellulosic materials, such as food waste, sugarcane bagasse and paper mill waste [4e6], have been studied extensively. Grass silage is a potential cellulosic material for biological hydrogen production. ...
... methanogens. Methanogens can be inhibited, for example by heat-treatment [3] or chemicals [6] to increase hydrogen yields. Increasing substrate concentration has been reported to increase the hydrogen production rates [13] [14] but to decrease hydrogen yields [15]. ...
... Duplicate samples were taken aseptically from the end points of hydrogen production experiments, samples were centrifuged (7500 g, 10 min) and the biomass was stored at À20 C. DNA was extracted with a PowerSoilä DNA isolation kit (MoBio Laboratories, Inc.). PCR-DGGE analysis, the reamplification of the bands as well as analysis of the sequence data were done as described by Nissilä et al. [3] ...
In this study, grass silage was used both as a source of bacteria and as a substrate for dark fermentative hydrogen production. Silage is produced by lactic acid fermentation controlled by end point pH (<4.0). In this study, the fermentation of silage was successfully continued and directed to hydrogen production by neutralizing the pH. Highest hydrogen yield of 37.8 ± 5.8 mL H2/g silage was obtained at 25 g/L of silage. The main soluble metabolites were acetate and butyrate with the final concentrations of 1.5 ± 0.2 and 0.5 ± 0.0 g/L, respectively. Bacteria present (at 25 g silage/L) included Ruminobacillus xylanolyticum, Acetanaerobacterium elongatum and Clostridium populeti and were involved in silage fermentation to hydrogen. In summary, this work demonstrates that grass silage becomes amenable to hydrogen fermentation by indigenous silage bacteria through pH neutralization.
... Hydrogen gas has a high-energy content (122 MJ/kg) [11] and produces only water when combusted [12]. The enrichment procedure for hydrogen producing communities often involves pretreatment, such as heat, acid or base, to inhibit methanogens [13][14][15][16]. However, some pretreatments may also inhibit some cellulolytic and hydrogen producing bacteria [14,15,17]. ...
... Thus, with complex substrates pretreatments cannot always be used [18]. High hydrogen yields have also been reported with non-pretreated cultures [16,19]. Fermentation of food waste (initial concentration 8 g VS/ L/d) resulted in 90% cellulose degradation and a hydrogen yield of 2.2 mol H 2 /mol hexose [18]. ...
... Fermentation of food waste (initial concentration 8 g VS/ L/d) resulted in 90% cellulose degradation and a hydrogen yield of 2.2 mol H 2 /mol hexose [18]. In our previous study, an enriched compost culture degraded 57% of the cellulose (initial concentration 5 g/L) and produced hydrogen with a yield of 2.4 mol H 2 /mol hexose degraded [16]. ...
Conifer and birch pulp fermentation to hydrogen and methane was studied using dry and wet pulps with a compost enrichment culture at a pH range from 6 to 9. Hydrogen was produced at each pH, whilst methane was produced at all other pH values except pH 6 with dry conifer pulp and pH 9. Hydrogen and methane yields were generally higher with birch than with conifer pulp and the overall energy yields were higher with wet than dry pulp. The highest hydrogen and methane yields were 560 mL H2/g TS with wet birch pulp at pH 6 and 4800 mL CH4/g TS with wet conifer pulp at pH 7, respectively. Fermentation of dry pulps at pH 6 resulted in 160 mL H2/g TS. Hydrogenic bacteria belonging to phyla Bacteroidetes, Firmicutes and Proteobacteria were present in the cultures. Hydrogen was also produced from chemically hydrolyzed pulps. The highest hydrogen yield from dry conifer pulp hydrolysate was 63 mL H2/g TS. In summary, hydrogen and energy (calculated as H2) yields were higher with direct fermentation than from chemically hydrolyzed pulps. However, chemical hydrolysis followed by hydrogen production required less than 10 days compared to 28 days required for direct pulp fermentation to hydrogen.
... The treatments with G. mosseae inoculation and/or compost addition affected a number of bacterial orders in the soils, including Acidobacteriales, Actinomycetales, Clostridiales, Sphingomonadales, Cytophagales, Gemmatimonadales, Xanthomonadales and Saprospirales. The similar phenomena were also observed in early studies (Nissilä et al., 2011;Schmalenberger et al., 2013;Hervé et al., 2014;Shabarova et al., 2014;Krustok et al., 2015). The Actinomycetales and Xanthomonadales were significantly abundant in the treatments with compost and G. mosseae, representing over the one fourth of total microbial communities (Fig. 3). ...
... Our results also showed that the bacteria in the orders of Actinomycetale, Clostridiales, Sphingomonadales and Cytophagales enriched in the treatments with compost (Fig. 3). Nissilä et al. (2011) revealed that Clostridiaceae (Clostridiales) was mainly responsible for hydrogen production and cellulose degradation in compost-enrichment cultures. Clostridiales and Actinomycetales could both contribute to the formation of communities in the thermophilic stages of composting and secrete bacterial enzymes to enhance deconstruction of recalcitrant lignocellulose during composting process (Martins et al., 2013). ...
An experiment was conducted to improve rhizoremediation for decabromodiphenyl ether (BDE-209) contaminated soil from typical E-waste dismantling areas. Plants of ryegrass (Lolium perenne L.) and rice (Oryza sativa L.) were cultivated in aged-contaminated (initial concentration of 346.3 μg BDE-209$kg⁻¹) and freshly-spiked (initial concentration of 3127 μg BDE-209$kg⁻¹) soils, coupling with the agricultural modification strategies of compost addition and/or arbuscular mycorrhizal fungi (AMF) infection, respectively. 60 days’ growth of ryegrass significantly facilitated the dissipation of BDE-209, with the most effective in its rhizosphere in treatment inoculated with AMF; the BDE-209 dissipation rates achieved 51.9% and 22.8% in rhizosphere, and 43.5% and 19.8% in non-rhizosphere, for aged-contaminated and freshly-spiked soils, respectively. 120 days’ growth of rice with simultaneous inoculation of AMF and addition of compost was the most effective in facilitating BDE-209 dissipation in aged-contaminated soil, with the removal rates of 53.3% and 48.1% in rhizosphere and non-rhizosphere soils respectively; while for freshly-spiked soils, the most effective removal was achieved by compost addition only, with the BDE-209 dissipation rates of 27.9% and 26.6% in rhizosphere and non-rhizosphere soils, respectively. High throughput sequencing analysis of rhizosphere soil DNA showed that responses in microbial communities and their structure differed with plant species, soil pollution dose, AMF inoculation and/or compost addition. Actinomycetales, Xanthomonadales, Burkholderiales, Sphingomonadales, Clostridiales, Cytophagales, Gemmatimonadales and Saprospirales were the sensitive responders and even possibly potential functional microbial groups during the facilitated removal of BDE-209 in soils. This study illustrates an effective rhizoremediation pattern for removal of BDE-209 in pollution soils, through successive cultivation of rice and followed by ryegrass, with rice growth coupled with AMF inoculation and compost addition, while ryegrass growth coupled with AMF inoculation only.
... The currently reported cellulose bioconversion efficiency was in a range of 40-90 % (Cheng and Liu 2012;Nissila et al. 2011;Geng et al. 2010;Forrest et al. 2011). In this study, after 200 days' enrichment, the cellulose conversion capacity of the mesophilic consortium ( Figure S1) was enhanced significantly from 12 to 70 %, and this conversion ratio was slightly lower than that of 85 % at the thermophilic temperature in our previous study (Xia et al. 2012). ...
... More detailed and quantitative information on these classes and families was summarized in Table S5 and Table S6, respectively. Based on the previous assumption that the enriched taxa might be those that were active in the cellulose bioconversion process and also based on some literature review, the proposed functional roles of the enriched families are summarized in Fig. 3. Clostridiaceae and Ruminococcaceae were the two cellulose-hydrolyzing families (Nissila et al. 2011;Wu and He 2013) enriched in both the mesophilic and the thermophilic consortia, while Opitutaceae and Spirochaetaceae were the cellulose hydrolysers (Rodrigues and Isanapong 2014;Karami et al. 2014) specifically enriched in the mesophilic consortium and the thermophilic consortium, respectively. In consistent with the higher propionate concentration detected in the mesophilic cellulose conversion process, enrichment of Propionibacteriaceae, the taxa capable in propionate generation from lactate (Falentin et al. 2010), was observed in the mesophilic consortium. ...
Improvement on the bioconversion of cellulosic biomass depends much on the expanded knowledge on the underlying microbial structure and the relevant genetic information. In this study, metagenomic analysis was applied to characterize an enriched mesophilic cellulose-converting consortium, to explore its cellulose-hydrolyzing genes, and to discern genes involved in methanogenesis. Cellulose conversion efficiency of the mesophilic consortium enriched in this study was around 70 %. Apart from methane, acetate was the major fermentation product in the liquid phase, while propionate and butyrate were also detected at relatively high concentrations. With the intention to uncover the biological factors that might shape the varying cellulose conversion efficiency at different temperatures, results of this mesophilic consortium were then compared with that of a previously reported thermophilic cellulose-converting consortium. It was found that the mesophilic consortium harbored a larger pool of putative carbohydrate-active genes, with 813 of them in 54 GH modules and 607 genes in 13 CBM modules. Methanobacteriaceae and Methanosaetaceae were the two methanogen families identified, with a preponderance of the hydrogenotrophic Methanobacteriaceae. In contrast to its relatively high diversity and high abundance of carbohydrate-active genes, the abundance of genes involved in the methane metabolism was comparatively lower in the mesophilic consortium. A biological enhancement on the methanogenic process might serve as an effective option for the improvement of the cellulose bioconversion at mesophilic temperature.
... Most clostridia species that are not direct hydrogen producers have been left out. However, they may be indirectly responsible for hydrogen production by degradation of cellulose materials (e.g., Clostridium cellulosi and Clostridium stercorarium) (Hung et al. 2011a;Nissilä et al. 2011). ...
... Also, there are reports of a cocultured bacteria in hydrogen production from Bifidobacterium sp. that helps in breaking down starch into smaller molecules making it convenient for utilization by Clostridium species (Cheng et al. 2008). There are several other examples existing for this strategy (Hung et al. 2011a;Nissilä et al. 2011). ...
Hydrogen gas exhibits potential as a sustainable fuel for the future. Therefore, many attempts have been made with the aim of producing high yields of hydrogen gas through renewable biological routes. Engineering of strains to enhance the production of hydrogen gas has been an active area of research for the past 2 decades. This includes overexpression of hydrogen-producing genes (native and heterologous), knockout of competitive pathways, creation of a new productive pathway, and creation of dual systems. Interestingly, genetic mutations in 2 different strains of the same species may not yield similar results. Similarly, 2 different studies on hydrogen productivities may differ largely for the same mutation and on the same species. Consequently, here we analyzed the effect of various genetic modifications on several species, considering a wide range of published data on hydrogen biosynthesis. This article includes a comprehensive metabolic engineering analysis of hydrogen-producing organisms, namely Escherichia coli, Clostridium, and Enterobacter species, and in addition, a short discussion on thermophilic and halophilic organisms. Also, apart from single-culture utilization, dual systems of various organisms and associated developments have been discussed, which are considered potential future targets for economical hydrogen production. Additionally, an indirect contribution towards hydrogen production has been reviewed for associated species.
... The genus Thermoclostridium, which is also more abundant in the control treatment, includes two known anaerobic species, T. caenicola and T. stercorarium. Their cellulolytic potential is widely recognised and they have been observed in several composting sites (Madden, 1983;Nissilä et al., 2011), but no studies have mentioned a propensity of these species to be sensitive to glyphosate. Finally, ASVs identified as the genera Pseudomonas and Streptomyces were also more abundant in the control than in the glyphosate treatment. ...
The herbicide glyphosate has several potential entry points into composting sites and its impact on composting processes has not yet been evaluated. To assess its impact on bacterial diversity and abundance as well as on community composition and dynamics, we conducted a mesocosm experiment at the Montreal Botanical Garden. Glyphosate had no effect on physi-cochemical property evolution during composting, while it was completely dissipated by the end of the experiment. Sampling at Days 0, 2, 28 and 112 of the process followed by 16S rRNA amplicon sequencing also found no effect of glyphosate on species richness and community composition. Differential abundance analyses revealed an increase of a few taxa in the presence of glyphosate, namely TRA3-20 (order Polyangiales), Pedo-sphaeraceae and BIrii41 (order Burkholderiales) after 28 days. In addition, five amplicon sequence variants (ASVs) had lower relative abundance in the glyphosate treatment compared to the control on Day 2, namely Coma-monadaceae, Pseudomonas sp., Streptomyces sp., Thermoclostridium sp. and Actinomadura keratinilytica, while two ASVs were less abundant on Day 112, namely Pedomicrobium sp. and Pseudorhodoplanes sp. Most differences in abundance were measured between the different sampling points within each treatment. These results present glyphosate as a poor determinant of species recruitment during composting.
... The volume of the gas (H 2 and CO 2 ) produced in the BHP assays was quantified every day by syringe method [23]. The gas constituents were analysed with a Shimadzu gas chromatograph (GC-2014) with a thermal conductivity detector according to Nissilä et al. [24]. Cumulative H 2 production was determined using the mass balance equation of Logan et al. [25], after the hydrogen or methane content of the controls was excluded. ...
The possibility of producing hydrogen and methane from sedimented pulp and paper mill waste fibre was explored for the first time in a double-stage process. Hydrogen and methane production was compared in batch experiments under four different conditions: two-stage hydrogen and methane production under (i) mesophilic (37 °C) and (ii) thermophilic (55 °C) and one-stage methane production under (iii) mesophilic and (iv) thermophilic conditions. Among these conditions studied, two-stage thermophilic anaerobic digestion achieved the highest hydrogen yield (42.1 ± 2.91 mL/g VS) and methane yield (334 ± 26.8 mL/g VS) at 55 °C. The experimental results were fitted to modified Gompertz equation, and a strong correlation was built from the overall magnitude of the regression (R² ranged from 0.996to 0.989) between the experimental data and the applied equation. Total energy yield from the two-stage thermophilic process was higher (3.7 kWh/L) than the one-stage process (1.7 kWh/L). The two-stage treatment also reduced the treatment time by half. Knowledge gained from this study will provide a basis for future investigation of two-stage treatment of sedimented fibres.
Graphical abstract
... They are capable decomposing large molecular organic compounds (primarily cellulose and pectins), to produce hydrogen and methane. The most often mentioned in this context are anaerobic bacteria of Thermoanaerobacteriaceae and Clostridiaceae families (Nissilä et al. 2011), as well as of Methanosarcina, Methanosaeta, Methanobrevibacter, and Methanobacterium genera (Ike et al. 2010). Among these groups a special position has Clostridium genus, of which about 35 species can cause clinically important adverse effects, mainly due to the release of their toxins (Samul et al. 2013). ...
The study focused on exposure assessment to bacterial aerosols and organic dust in waste sorting plant. Samples were collected at different workplaces of waste sorting cycle i.e.: waste press, reloading area, loading of conveyor belt, sorting cabin, sorting hall, and control room. A quantitative analysis of aerobic and anaerobic bacteria was supplemented by qualitative analysis of anaerobic biota with the use of culture-based methods and biochemical tests. In addition, inhalable dust concentrations were also evaluated. To confirm the presence of Clostridium genus, the PCR reaction with specific primers (Chis150f and ClostIr) was performed. The average concentration of total bacteria in waste sorting plant was 4347 CFU m⁻³ (SD = 2439), of which 66% were anaerobic strains (2852 CFU m⁻³; SD = 2127). It was found that about 24% of anaerobic bacteria belonged to Clostridium genus (682 CFU m⁻³; SD = 633). The highest contamination with anaerobic bacteria was observed near the waste reloading plant (3740 CFU m⁻³), and the lowest in the control room (850 CFU m⁻³). The average concentration of inhalable dust in the waste sorting plant was 0.81 mg m⁻³ (SD = 0.59). The correlation analysis showed that the presence of anaerobic bacteria, including clostridia was significantly determined by the microclimate parameters. Qualitative analysis showed the presence of 16 anaerobic species belonging to 9 genera, of which Actinomyces, Clostridium and Gemella were present at all workplaces. The molecular analysis confirmed the presence of Clostridium genus in both bioaerosol and settled dust samples.
Implications
The study showed that anaerobic bacteria should be taken into account as an important component of this microbiota when assessing the exposure of waste sorting workers to biological agents. However, future studies should investigate more precisely how the composition of sorted waste as well as the season can affect the diversity of anaerobic bacteria in this working environment. More attention should be paid to regular cleaning of equipment surfaces in the plant, as deposited organic dust is an important reservoir of anaerobic bacteria, including those of a potentially pathogenic nature.
... Notamment, les substrats les plus utilisés pour produire de l'hydrogène sont : le glucose (Lin & Chang, 1999 ;Van Ginkel & Logan, 2005b ;Li et al., 2008 ;Quéméneur et al., 2010) ; le saccharose (Chen et al., 2001 ;Chen & Lin, 2003 ;Lo et al., 2008 ;Mariakakis et al., 2011) et la cellulose (Ueno et al., 1995 ;Nissilä et al., 2011). Enfin, il a été démontré que, dans une gamme appropriée, l'augmentation de la concentration en substrat pouvait améliorer les performances de production d'hydrogène (Wang & Wan, 2009a). ...
Les cultures mixtes sont aujourd’hui considérées comme une sérieuse alternative aux cultures pures dans le cadre des biotechnologies du fait leur capacité à traiter une large variété de substrats organiques et ce en conditions non stériles. La principale restriction à leur utilisation réside toutefois dans une instabilité du procédé liée à la présence de voies métaboliques non désirées résultant d’interactions microbiennes complexes. Notamment, le rôle des bactéries de faible abondance dans les écosystèmes reste à être élucidé. Ce travail a donc consisté à déterminer le rôle des bactéries minoritaires dans la production d’hydrogène par voie fermentaire. Dans un premier temps, sept inocula ont été cultivés en réacteurs continus, dans les mêmes conditions opératoires. La même espèce dominante a été observée six fois sur sept mais les performances de production d’hydrogène différaient. Seules la nature et la diversité des espèces minoritaires variaient d’un écosystème à l’autre prouvant ainsi que les bactéries en proportion minoritaires jouent un rôle clé en orientant le métabolisme global de l’écosystème. Dans un second temps, certaines de ces espèces minoritaires ont été utilisées comme perturbateurs biotiques. Pour cela, un écosystème producteur d’hydrogène a été modifié artificiellement en introduisant des souches bactériennes exogènes aux fonctions redondantes et/ou complémentaires des souches indigènes. Les résultats en réacteur batch ont montré que les performances de production d’hydrogène pouvaient ainsi être améliorées. Globalement, les résultats obtenus ne peuvent être expliqués par de simples interactions trophiques et suggèrent la présence de mécanismes d’interactions de coopération entre microorganismes. De plus, sous des conditions opératoires plus favorables, l’insertion de certaines espèces minoritaires a permis de stabiliser l’écosystème microbien, sans pour autant en affecter la production d’hydrogène. Dans tous les cas, les interactions compétitives n'ont pas été favorables à la production d'hydrogène. Enfin, des essais en réacteur continu ont montré que le mode d’implantation des souches peut être un facteur primordial.
... Gas produced in the CSTRs was measured by water displacement method every working day. The gas composition was analyzed with a Shimadzu gas chromatograph (GC-2014) equipped with a Porapak N column (80/100 mesh) and a thermal conductivity detector according to Nissilä et al. [30]. Cumulative H 2 and CO 2 production were calculated according to Logan et al. [31]. ...
The high volumes of sewage sludge produced have raised interests for simultaneous treatment and clean energy production, e.g. in the form of hydrogen. Pretreatment of sewage sludge is required to enhance microbial degradation and in turn hydrogen yield from sewage sludge. The potential of five substrate pretreatments, individually and in combinations, to increase biohydrogen production from mixed primary and secondary sewage sludge at four incubation pH (5, 7, 9, and 11) was studied in batch assays. Alkali + ultrasonication pretreatment increased the hydrogen production almost seven times (0.35 mmol H2/g VS) compared to untreated sewage sludge at initial pH 11. In general, higher hydrogen yields and lower acetate concentrations were obtained under alkaline conditions (pH 9 and 11), being more favorable for protein degradation and not favorable for hydrogen consumption via homoacetogenesis. Subsequently, fermentation of alkali + ultrasonication pretreated sewage sludge in a semi-continuous stirred tank reactor (CSTR) produced a maximum hydrogen yield of 0.1 mmol H2/g VS, three times higher than the yield obtained from alkali pretreated sludge. The gas produced in the CSTRs contained a low concentration of CO2 (< 5%), and is thus easily upgradable to biohydrogen.
Graphic Abstract
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... The lowest methane production in R2i, justifies the highest hydrogen production observed in this condition. The hydrogen consumption by methanogenic archaea is a disadvantage of using microbial consortium as inoculum source [67] when hydrogen is the end target product. In this study, the presence of methanogenic archaea such as Methanothermobacter genus in the thermophilic sludge results in hydrogen consumption for methane conversion. ...
The goals of this study were to describe a comprehensive taxonomic profile of bacterial communities endogenous from sugarcane bagasse (SCB) and from a thermophilic sludge formed mainly by Proteobacteria Actinobacteria and Firmicutes phylum and its potential as a bioaugmented inoculum for degradation of lignocellulosic biomass. Batch assays were performed using SCB as substrate at different condition: (RC) 2 g L−1 glucose, (R1i) 2 g L−1 unpretreated SCB, (R2i) 2 g L−1 hydrothermally pretreated SCB (at 210 °C for 15 min), (R3i) 2 g L−1 hydrothermally pretreated SCB (at 210 °C for 15 min) followed by alkaline delignification (NaOH—1 M), (R4i) 1 g L−1 unpretreated SCB plus 1 g L−1 hydrothermally pretreated SCB (at 210 °C for 15 min) followed by alkaline delignification. Hydrogen, methane and organic acids were the main metabolites produced during the fermentation. Maximum hydrogen (2.01 and 1.09 mol H2 mol−1 consumed soluble carbohydrates) were obtained in R1i and R2i, respectively. The highest organic acid (1051 mg L−1) and methane (0.92 mmo L−1) production were obtained in R4i.
... Similarly, O-thong et al. successfully isolated Thermoanaerobacterium thermosaccharolyticum PSU-2 from POME in Thailand and showed a high H 2 yield of 1.3 mol H 2 /mol hexose [85], which was slightly lower than the yield observed in this study (1.6 mol H 2 /mol hexose from 80% POME in batch fermentation). This particular bacterium was found to have the capability of degrading complex carbohydrates such as starch and cellulosic materials and utilising different kinds of carbon sources [81][82][83][84][85][86][87][88][89][90][91][92], which indicates its versatility and tolerance for practical applications [93]. In our study, an efficient dark fermentation was carried out to generate good H 2 gas production by attached biofilm by utilising both glucose and xylose from mimicking sugar solution and in POME wastewater fermentation. ...
In this study, we examined the formation of thermophilic microbial biofilm by self-attachment on microbial carrier of granular activated carbon (GAC) in five different micro-pore volumes 0.31, 0.41, 0.44, 0.48, and 0.50 cm³/g. It was found that the highest hydrogen production rate of 100.8 ± 3.7 mmol H2/l.d and yield of 1.01 ± 0.07 mol H2/mol sugar were obtained at 0.44 cm³/g volume size of GAC. The cellulolytic activity of attached-biofilm was further investigated using POME as a feedstock. The results showed that in all diluted POME substrate, the total sugar consumed by the microbes was found higher than that the amount of soluble monomeric sugar present in the POME medium. It is believe that the microbial biofilm was able to hydrolyse polymeric sugar of cellulosic fibre in the POME by performing enzymatic hydrolysis into simple monomeric sugar. The isolated biofilm bacteria that subjected to 16S rRNA gene analysis presented 99% high homology to the species of Thermoanaerobacterium thermosaccharolyticum which were guaranteed to perform a cellulosic degradation activity.
... For example, Coprococcus, Oxobecter, and Ruminiclostridium revealed significant negative correlations with the concentration of PCP in the reactors (Fig. S6 in SI), but there was no direct evidence thus far about their involvement in PCP degradation. Coprococcus and Ruminiclostridium have always been reported to produce acetate via fermentative metabolisms (Tsai and Jones 1975;Patel et al. 1981;Nissila et al. 2011;Zhang et al. 2014). The presence of this metabolic product might enhance the degradation rate of PCP (Chang et al. 1995). ...
Pentachlorophenol (PCP) is a common persistent pesticide in soil that has generated a significant environmental problem worldwide. Therefore, anaerobic degradation of PCP by the soil indigenous microbial community has gained increasing attention. However, little information is available concerning the functional microorganisms and the potential shifts in the microbial community associated with PCP degradation. In this study, we conducted a set of experiments to determine which components of the indigenous microbial community were capable of degrading PCP in soils of two land use types (upland and paddy soils) in southern China. Our results showed that the PCP degradation rate was significantly higher in paddy soils than that in upland soils. 16S ribosomal RNA (rRNA) high-throughput sequencing revealed significant differences in microbial taxonomic composition between the soil with PCP and blank (soil without PCP) with Acinetobacter, Clostridium, Coprococcus, Oxobacter, and Sedimentibacter dominating the PCP-affected communities. Acinetobacter was also apparently enriched in the paddy soils with PCP (up to 52.2 %) indicated this genus is likely to play an important role in PCP degradation. Additionally, the Fe(III)-reducing bacteria Clostridium may also be involved in PCP degradation. Our data further revealed hitherto unknown metabolisms of potential PCP degradation by microorganisms including Coprococcus, Oxobacter, and Ruminiclostridium. Overall, these findings indicated that land use types may affect the PCP anaerobic degradation rate via the activities of indigenous bacterial populations and extend our knowledge of the bacterial populations responsible for PCP degradation.
... Moreover, the diverse microflora present in the seed sludge might provide synergistic interactions that improve substrate degradation and thus enhance H 2 production [72]. Anaerobic sludge [73], sewage sludge [74], and compost [75] have been used as mixed inoculum for fermentative hydrogen production. But the microflora in the seed sludge usually consists of both H 2consuming and H 2 -producing bacteria. ...
Hydrogen is a promising alternative to fossil fuel for a source of clean energy. Thermophilic biohydrogen production is beneficial for obtaining high H2 production yield. This review recapitulates the basic metabolic pathways in bacteria for hydrogen production and the enzymes involved in various thermophilic hydrogen producing pathways in microorganisms. It also focuses on the current status of thermophilic biohydrogen production through fermentation of commercially viable substrates, such as agricultural residues. The use of metabolic engineering to attain certain physiological desirable characteristics in H2-producing microorganisms, culture conditions, and types of bioreactors to be used are reviewed. Major obstacles in industrial production of biohydrogen like low volumetric hydrogen production and its environmental impact are identified. The review has further identified current limitations in the commercial thermophilic hydrogen production and suggested methods like the use of heat exchangers and effluent recirculation to reduce the production cost.
... Coprothermobacter was selected during thermophilic dry anaerobic digestion of model garbage with ammonia stripping (Yabu et al. 2011) and found in thermophilic dry methanogenic systems fed with garbage stillage (Tang et al. 2011). Kawagoshi et al. (2005) identified Coprothermobacter by DGGE in a hydrogen-producing reactor inoculated with six different sources and treating a synthetic medium, while Nissilä et al. (2011) detected Coprothermobacter in a batch enrichment of hydrogen-producing, cellulolytic cultures. Coprothermobacter was found at the biocathode during hydrogen production in a two-chambered microbial electrolysis cells, representing 19.8% of the total clones (Fu et al. 2013c). ...
Thermophilic bacteria have been isolated from several terrestrial, marine and industrial environments. Anaerobic digesters treating organic wastes are often an important source of these microorganisms, which catalyze a wide array of metabolic processes. Moreover, organic wastes are primarily composed of proteins, whose degradation is often incomplete. Coprothermobacter spp. are proteolytic anaerobic thermophilic microbes identified in several studies focused on the analysis of the microbial community structure in anaerobic thermophilic reactors. They are currently classified in the phylum Firmicutes; nevertheless, several authors showed that the Coprothermobacter group is most closely related to the phyla Dictyoglomi and Thermotoga. Since only a few proteolytic anaerobic thermophiles have been characterized so far, this microorganism has attracted the attention of researchers for its potential applications with high temperature environments. In addition to proteolysis, Coprothermobacter spp. showed several metabolic abilities and may have a biotechnological application either as source of thermostable enzymes or as inoculum in anaerobic processes. Moreover, they can improve protein degradation by establishing a syntrophy with hydrogenotrophic archaea. To gain a better understanding of the phylogenesis, metabolic capabilities and adaptations of these microorganisms it is of importance to better define the role in thermophilic environments and to disclose properties not yet investigated.
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... The strategy for multiclass analysis was all-against-all. Partial least squares (PLS) regression was performed with JMP 11.0.0 (SAS Institute, Inc.) to assess the relation between the gut microbiota composition and host phenotypes (28). The predictors and responses were centered and scaled to have mean 0 and SD 1. ...
Background: Whole grain consumption reduces the risk of major chronic diseases. It is not clear how whole grains exert their beneficial effects.
Objective: The aim was to compare the physiologic effects of whole grain oat (WGO) flour with low bran oat (LBO) flour.
Methods: Two AIN-93G-based diets were formulated with either WGO or LBO flour. Five-week-old male C57BL/6J mice were fed LBO (n = 11) and WGO (n = 13) diets for 8 wk. Cecal microbiota was profiled by pyrosequencing of the 16S ribosomal RNA gene. Data are reported as means ± SEMs or antilogs of the mean (mean − SEM, mean + SEM).
Results: The weight gain was 14.6% less in the WGO group during week 7 (P = 0.04). WGO improved insulin sensitivity as reflected by significantly lower plasma insulin [1500 (1370, 1650) ng/L vs. 2340 (2090, 2620) ng/L; P = 0.006], C-peptide (3980 ± 548 ng/L vs. 7340 ± 1050 ng/L; P = 0.007), and homeostasis model assessment-estimated insulin resistance (21.4 ± 2.3 vs. 34.7 ± 4.9; P = 0.03). Plasma total cholesterol was 9.9% less and non-HDL cholesterol was 11% less in the WGO group. A comparison of relative abundance indicated Prevotellaceae, Lactobacillaceae, and Alcaligenaceae families were 175.5% (P = 0.03), 184.5% (P = 0.01), and 150.0% (P = 0.004), respectively, greater in the WGO group and Clostridiaceae and Lachnospiraceae families were 527% (P = 0.004) and 62.6% (P = 0.01), respectively, greater in the LBO group. Cecal microbiota composition predicts 63.9% variation in plasma insulin and 88.9% variation in plasma non-HDL cholesterol.
Conclusions: In mice, WGOs improved insulin sensitivity and plasma cholesterol profile compared with LBOs, and the effects were associated with the changes in cecal microbiota composition. Increasing WGO consumption may help improve insulin sensitivity and dyslipidemia in chronic diseases.
Developing novel strategies to enhance volatile fatty acid (VFA) yield from abundant waste resources is imperative to improve the competitiveness of biobased VFAs over petrochemical-based VFAs. This study hypothesized to improve the VFA yield from food waste via three strategies, viz., pH adjustment (5 and 10), supplementation of selenium (Se) oxyanions, and heat treatment of the inoculum (at 85 °C for 1 h). The highest VFA yield of 0.516 g COD/g VS was achieved at alkaline pH, which was 45% higher than the maximum VFA production at acidic pH. Heat treatment resulted in VFA accumulation after day 10 upon alkaline pretreatment. Se oxyanions acted as chemical inhibitors to improve the VFA yield at pH 10 with non-heat-treated inoculum (NHT). Acetic and propionic acid production was dominant at alkaline pH (NHT); however, the VFA composition diversified under the other tested conditions. More than 95% Se removal was achieved on day 1 under all the conditions tested. However, the heat treatment was detrimental for selenate reduction, with less than 15% Se removal after 20 days. Biosynthesized Se nanoparticles were confirmed by transmission and scanning electron microscopy and and energy dispersive X-ray analyses. The heat treatment inhibited the presence of nonsporulating bacteria and methanogenic archaea (Methanobacteriaceae). High-throughput sequencing also revealed higher relative abundances of the bacterial families (such as Clostridiaceae, Bacteroidaceae, and Prevotellaceae) that are capable of VFA production and/or selenium reduction.
Under the requirements of China's strategic goal of "carbon peaking and carbon neutrality", as a renewable, clean and efficient secondary energy source, hydrogen benefits from abundant resources, a wide variety of sources, a high combustion calorific value, clean and non-polluting, various forms of utilization, energy storage mediums and good security, etc. It will become a realistic way to help energy, transportation, petrochemical and other fields to achieve deep decarbonization, and will turn into an important replacement energy source for China to build a modern clean energy system. It is clear that accelerating the development of hydrogen energy has become a global consensus. In order to provide a theoretical support for the accelerated transformation of hydrogen-related industries and energy companies, and provide a basis and reference for the construction of "Hydrogen Energy China", this paper describes main key technological progresses in the hydrogen industry chain such as hydrogen production, storage, transportation, and application. The status and development trends of hydrogen industrialization are analyzed, and then the challenges faced by the development of the hydrogen industry are discussed. At last, the development and future of the hydrogen industry are prospected. The following conclusions are achieved. (1) Hydrogen technologies of our country will become mature and enter the road of industrialization. The whole industry chain system of the hydrogen industry is gradually being formed, and will realize the leap-forward development from gray hydrogen, blue hydrogen to green hydrogen. (2) The overall development of the entire hydrogen industry chain such as hydrogen production, storage and transportation, fuel cells, hydrogen refueling stations and other scenarios should be accelerated. Besides, in-depth integration and coordination with the oil and gas industry needs more attention, which will rapidly promote the high-quality development of the hydrogen industry system. (3) The promotion and implementation of major projects such as "north-east hydrogen transmission", "west-east hydrogen transmission", "sea hydrogen landing", and utilization of infrastructures such as gas filling stations, can give full play to the innate advantages of oil and gas companies in industrial chain nodes such as hydrogen production and refueling, etc., which can help to achieve the application of "oil, gas, hydrogen, and electricity" four-station joint construction, form a nationwide hydrogen resource guarantee system, and accelerate the planning and promotion of the "Hydrogen Energy China" strategy.
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 growing global population has placed a large burden on the agricultural sector to increase food production. At the same time, concern for the environment in regards to water pollution, greenhouse gas emission, and non-renewable energy usage, is increasing. Anaerobic digestion (AD) offers an environmentally friendly method to treat agricultural wastes while producing renewable bioenergy. One derivation of the AD process is biohydrogen production through fermentation of organic wastes. During the anaerobic degradation of organic matter, hydrogen is produced as a by-product of the conversion of complex oligomers to volatile fatty acids. Hydrogen has an energy density of 122 kJ g-1 compared to 43 kJ g-1 for gasoline, and the only product of its combustion is water. As such, hydrogen has great potential as an energy carrier. Although biohydrogen can be produced from organic materials via fermentation, the efficiency of this process is low due to thermodynamic constraints and metabolic limits. Engineering the fermentation process can alleviate some of the problems causing low efficiency production. Manipulation of fermentation conditions, inoculum composition, substrate composition, and separation of the anaerobic degradation process into multiple stages, either physically or temporally, can all be used to enhance biohydrogen production. The potential to couple fermentative biohydrogen production with conventional methane producing AD processes further enhances prospective energy gains from organic agricultural wastes.
The prevalence of lactic acid bacteria (LAB) and the effects of their antimicrobial peptides on H2 production in anaerobic fluidized bed reactors (AFBRs) operated with cheese whey (AFBR1 and AFBR2) were verified in this study. The AFBR1 received 5 g COD L−1 of cheese whey with decreasing hydraulic retention times (HRT) of 14 to 8 h. The AFBR2 was operated with 3-10 g COD L−1 of cheese whey with an HRT of 6 h. Next, 152 colonies were selected from de Man, Rogosa and Sharpe MRS agar plates, and 45 strains were classified as LAB. The counts oscillated between 6.6 and 8.1 log CFU mL−1, indicating that the LAB survived and persisted in the AFBRs. Pure cultures were identified using 16S rRNA gene sequencing, and Lactococcus lactis was the prevalent LAB (70%) in both reactors. The highest H2 yields (1.9 and 2.3 mol H2 mol lactose−1) were obtained during the first operational phase in both reactors when a low organic loading rate (OLR) was applied, and when the growth of Lactococcus spp. was associated with Leuconostoc pseudomesenteroides. The bacteriocin-producing LAB (mostly Lactobacillus spp.) found on the specific phases of reactors AFBR1 and AFBR2 exerted a remarkable influence on H2 yield.
Biohydrogen production from dark fermentation of lignocellulosic materials represents a huge potential in terms of renewable energy exploitation. However, the low hydrogen yield is currently hindering its development on industrial scale. This study reviewed various technologies that have been investigated for enhancing dark fermentative biohydrogen production. The pre-treatment technologies can be classified based on their applications as inoculum or substrates pre-treatment or they can be categorised into physical, chemical, physicochemical and biological based on the techniques used. From the different technologies reviewed, heat and acid pre-treatments are the most commonly studied technologies for both substrates and inoculum pre-treatment. Nevertheless, these two technologies need not necessarily be the most suitable since across different studies, a wide array of other emerging techniques as well as combined technologies have yielded positive findings. To date, there exists no perfect technology for either inoculum or substrate pre-treatment. Although the aim of inoculum pre-treatment is to suppress H2-consumers and enrich H2-producers, many sporulating H2-consumers survive the pre-treatment while some non-spore H2-producers are inhibited. Besides, several inoculum pre-treatment techniques are not effective in the long run and repeated pre-treatment may be required for continuous suppression of H2-consumers and sustained biohydrogen production. Furthermore, many technologies employed for substrates pre-treatment may yield inhibitory compounds that can eventually decrease biohydrogen production. Consequently, much research needs to be done to find out the best technology for both substrates and inoculum pre-treatment while also taking into consideration the energetic, economic and technical feasibility of implementing such a process on an industrial scale.
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Thermophilic bio-hydrogen production from xylan by anaerobic mixed cultures in elephant dung was conducted. The initial pH, temperature, and xylan concentration of 7.0, 55°C and 3.0 g/L, respectively, gave a respective maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 12.16 mmol H2/g xylan and 61.30 mL H2/L.d. The optimum conditions were used to produce hydrogen from sugarcane bagasse (SCB) in which an HY of 2.60 mmol H2/g SCB and a HPR of 59.78 mL H2/L.d were obtained. The hydrogen producers present in both xylan and SCB fermentation broth were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp.
Hydrogen production using cellulosic residues offers the possibility of waste minimization with renewable energy recovery. In the present study, heat-treated biomass purified from leachate was used as inoculum in batch reactors for hydrogen production fed with different concentrations of cellulose (2.5, 5.0 and 10 g/L), in the presence and absence of exogenous cellulase. The heat-treated biomass did not degrade cellulose and hydrogen production was not detected in the absence of cellulase. In reactors with cellulase, the hydrogen yields were 1.2, 0.6 and 2.3 mol H-2/mol of hydrolyzed cellulose with substrate degradation of 41.4, 28.4 and 44.7% for 2.5, 5.0 and 10 g/L cellulose, respectively. Hydrogen production potentials (P) varied from 19.9 to 125.9 mmol H-2 and maximum hydrogen production rates (R-m) were among 0.8-2.3 mmol H-2/h. The reactor containing 10 g/L of cellulose presented the highest P and R-m among the conditions tested. The main acid produced in reactors were butyric acid, followed by acetic, isobutyric and propionic acids. Bacteria similar to Clostridium sp. (98-99%) were identified in the reactors with cellulase. The heat-treated leachate can be used as an inoculum source for hydrogen production from hydrolyzed cellulose. Copyright
H2 production from cellulose, using rumen fluid as the inoculum, has been investigated in batch experiments. Methanogenic archaea were inhibited by acid pre-treatment, which also inhibited cellulolytic microorganisms, and in consequence, the conversion of cellulose to H2. Positive results were observed only with the addition of cellulase. H2 yields were 18.5 and 9.6 mmol H2 g cellulose(-1) for reactors with 2 and 4 g cellulose l(-1) and cellulase, respectively. H2 was primarily generated by the butyric acid pathway and this was followed by formation of acetic acid, ethanol and n-butanol. In reactors using 4 g cellulose l(-1) and cellulase, the accumulation of alcohols negatively affected the H2 yield, which changed the fermentation pathways to solventogenesis. PCR-DGGE analysis showed changes in the microbial communities. The phylogenetic affiliations of the bands of DGGE were 99 % similar to Clostridium sp.
Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H2/mol xylose. In CSTR the highest H2 yield and production rate at 45 °C were 1.97 mol H2/mol xylose and 7.3 mmol H2/h/L, respectively, and at 37 °C, 1.18 mol H2/mol xylose and 1.7 mmol H2/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H2 production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.
This paper reviews recent research developments in biological thermophilic lignocellulosic biomass conversion based on sixty four references published in the past 4 years (2009-2012). Bioconversion of hydrolysate and lignocellulosic biomass with or without pretreatment under thermophilic conditions (with temperatures higher than 50 degrees C) to fermentation products like hydrogen, methane, ethanol and carboxylic acids is discussed in terms of the bioaugmentation techniques and microorganisms involved. The discussion was divided into two branches according to the form of substrate applied: one branch targeted the fermentation of liquid hydrolysate (liquid fraction generated from the pretreatment of lignocellulosic biomass); the other one summarized the studies using raw or pretreated solid lignocellulosic biomass as a feedstock. Fermentation of the hydrolysate was discussed from the aspects of hydrolysate toxicity tolerance and hydrolysate detoxification techniques, while, process affecting parameters like pH, enrichment processes, substrate type and loading as well as microbial communities were reviewed for solid lignocellulosic biomass fermentation. Key information was compiled into four tables respectively summarizing the optimal fermentation conditions for the production of hydrogen/methane and ethanol/carboxylic acids from hydrolysate and lignocellulosic biomass. Information delivered in this article may shed light on the perspectives of the scientific and technical challenges faced by thermophilic anaerobic lignocellulose bioconversion.
Concentrated acid hydrolysis of cellulosic material results in high dissolution yields. In this study, the neutralization step of concentrated acid hydrolysate of conifer pulp was optimized. Dry conifer pulp hydrolysis with 55 % H(2)SO(4) at 45 °C for 2 h resulted in total sugar yields of 22.3-26.2 g/L. The neutralization step was optimized for solid Ca(OH)(2), liquid Ca(OH)(2) or solid CaO, mixing time, and water supplementation. The highest hydrogen yield of 1.75 mol H(2)/mol glucose was obtained with liquid Ca(OH)(2), while the use of solid Ca(OH)(2) or CaO inhibited hydrogen fermentation. Liquid Ca(OH)(2) removed sulfate to below 30 mg SO(4) (2-)/L. Further optimization of the neutralization conditions resulted in the yield of 2.26 mol H(2)/mol glucose.
Anaerobic fermentative biohydrogen production, the conversion of organic substances especially from organic wastes to hydrogen gas, has become a viable and promising means of producing sustainable energy. Successful biological hydrogen production depends on the overall performance (results of interactions) of bacterial communities, i.e., mixed cultures in reactors. Mixed cultures might provide useful combinations of metabolic pathways for the processing of complex waste material ingredients, thereby supporting the more efficient decomposition and hydrogenation of biomass than pure bacteria species would. Therefore, understanding the relationships between variations in microbial composition and hydrogen production efficiency is the first step in constructing more efficient hydrogen-producing consortia, especially when complex and non-sterilized organic wastes are used as feeding substrates. In this review, we describe recent discoveries on bacterial community composition obtained from dark fermentation biohydrogen production systems, with emphasis on the possible roles of microorganisms that co-exist with common hydrogen producers.
Over 160 publications related to fermentative hydrogen production from wastewater and solid wastes by mixed cultures are compiled and analyzed. Of the 98 reported cases, 57 used single substrates (mainly carbohydrates), 8 used actual wastewater, and 33 used solid wastes for hydrogen conversion. The key information is compiled in four tables: (1) pretreatment conditions for screening hydrogen-producing bacteria from anaerobic sludge or soil, and the process and performance parameters for (2) single substrates in synthetic wastewaters, (3) actual wastewaters, and (4) solid wastes. Process parameters discussed include pH, temperature, hydraulic retention time, seed sludge, nutrients, inhibitors, reactor design, and the means used for lowering hydrogen partial pressure. Performance parameters discussed include hydrogen yield, maximum volumetric production rate, maximum specific production rate, and conversion efficiency. The outlook for this new technology is discussed at the end.
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.
Hydrogen is a valuable gas as a clean energy source and as feedstock for some industries. Therefore, demand on hydrogen production has increased considerably in recent years. Electrolysis of water, steam reforming of hydrocarbons and auto-thermal processes are well-known methods for hydrogen gas production, but not cost-effective due to high energy requirements. Biological production of hydrogen gas has significant advantages over chemical methods. The major biological processes utilized for hydrogen gas production are bio-photolysis of water by algae, dark and photo-fermentation of organic materials, usually carbohydrates by bacteria. Sequential dark and photo-fermentation process is a rather new approach for bio-hydrogen production. One of the major problems in dark and photo-fermentative hydrogen production is the raw material cost. Carbohydrate rich, nitrogen deficient solid wastes such as cellulose and starch containing agricultural and food industry wastes and some food industry wastewaters such as cheese whey, olive mill and bakers yeast industry wastewaters can be used for hydrogen production by using suitable bio-process technologies. Utilization of aforementioned wastes for hydrogen production provides inexpensive energy generation with simultaneous waste treatment. This review article summarizes bio-hydrogen production from some waste materials. Types of potential waste materials, bio-processing strategies, microbial cultures to be used, bio-processing conditions and the recent developments are discussed with their relative advantages.
Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation are accomplished in a single process step by cellulolytic microorganisms. CBP offers the potential for lower biofuel production costs due to simpler feedstock processing, lower energy inputs, and higher conversion efficiencies than separate hydrolysis and fermentation processes, and is an economically attractive near-term goal for "third generation" biofuel production. In this review article, production of third generation biofuels from cellulosic feedstocks will be addressed in respect to the metabolism of cellulolytic bacteria and the development of strategies to increase biofuel yields through metabolic engineering.
Dark fermentation of microcrystalline cellulose to produce biohydrogen using mono-culture or co-culture of isolated strains was studied. A strain (X9)(X9) with high hydrogen yield from microcrystalline cellulose was isolated and identified to be closely affiliated with Clostridium acetobutylicum, ATCC 824. At 37∘C and pH 5.0, the mono-culture of X9X9 yields hydrogen with a 5-h time lag and end liquid products primarily of acetate and butyrate. The co-culture of X9X9 with another strain, Ethanoigenens harbinenseB49B49, which can produce hydrogen efficiently from monosaccharides but directly from microcrystalline cellulose, produced more efficiently the biohydrogen via ethanol-type fermentation metabolism compared with mono-culture X9X9 test. Bioaugmentation with X9+B49X9+B49 improved cellulose hydrolysis and subsequent hydrogen production rates as compared with that of mono-culture bioaugmentation with X9X9.
Techniques are presented for measuring the biodegradability (Biochemical Methane Potential—BMP) and toxicity (Anaerobic Toxicity Assay—ATA) of material subjected to anaerobic treatment. These relatively simple bioassays can be conducted in most research laboratories without the need for sophisticated equipment. BMP is a measure of substrate biodegradability determined by monitoring cumulative methane production from a sample which is anaerobically incubated in a chemically defined medium. The ATA measures the adverse effect of a compound on the rate of the total gas production from an easily-utilized, methanogenic substrate. These techniques are demonstrated by an analysis of the BMP and ATA of processed samples of peat.
ficiency on direct microbial assimilation of cellulosic materials. Considerable research efforts have been made to improve the pretreatment and hydrolysis of lignocellulosic materials. Development of novel and effective cellulase enzymes, optimization and improvement of cellulase system, as well as engineering approaches on cellulose pretreatment and saccharification are gaining increasing interest. Information from genomics and molecular genetics combined with improved genetic engineering offer a wide range of possibilities for enhancing performance of cellulose feedstock utilization and biohydrogen production. This study reviews key technologies and variables to be considered during biohydrogen production from lignocellulosic feedstock.
Clostridium stercorarium, a new species of anaerobic, sporeforming, thermophilic, saccharoclastic, cellulolytic bacteria, is described. The colonies produced by these bacteria on cellobiose agar are 3 to 6 mm in diameter, cream colored, glossy, and umbonate with fiat, entire margins. Single cells are straight rods 0.7 to 0.8 by 2.7 to 7.7 µm, with oval terminal spores. Fermentation products from cellulose include hydrogen, carbon dioxide, ethanol, acetate, and lactate. The deoxyribonucleic acid base composition of the type strain of C. stercorarium, NCIB 11754, is 39 mol% guanine plus cytosine, and its temperature optimum is 65%C. The specific epithet refers to the source of the original isolate, rotting vegetation (a compost heap).
Fermentations can be used to produce sustainable energy carriers, such as hydrogen and ethanol (EtOH), from biomass or organic waste materials. The aim of this research was to prospect efficient H 2 -and EtOH-producing thermophilic microorganisms derived from hot spring environments in Iceland. Hydrogen-and EtOH-producing enrichment cultures were obtained from various hot spring samples over a temperature range of 50–78 °C. The temperature dependencies for the most promising enrichments were determined with a temperature-gradient incubator. One of the enrichments (33HL) produced 2.10 mol of H 2 /mol of glucose at 59 °C. Another enrichment (9HG), dominated by bacteria closely affiliated with Thermoanaerobacter thermo-hydrosulfuricus, produced 0.68 mol of H 2 /mol of glucose, and 1.21 mol of EtOH/mol of glucose at 78 °C. Hydrogen and EtOH production by 9HG was characterized further in a continuous-flow bioreactor at 74 °C. The highest H 2 and EtOH yields of 9HG were obtained at pH 6.8 (0.3. Lactate production decreased the H 2 and EtOH yields in the continuous-flow bioreactor, and the yields were lower than those obtained in the batch fermentations. In conclusion, the thorough batch screening of Icelandic hot spring samples indicated some promising enrichments for H 2 or H 2 plus EtOH production from carbohydrate materials.
Adaptation of biohydrogen producing extreme-thermophilic bacteria to household solid waste (HSW) at extreme-thermophilic temperature (70 °C) was investigated. Inocula received from an extreme-thermophilic glucose fermentation reactor were exposed to increasing HSW concentrations from 1 g-VS/L to 10 g-VS/L via repeated batch cultivation. It was found that repeated batch cultivation was a very useful method to adapt and enrich biohydrogen producing mixed cultures that could ferment HSW with high hydrogen yield and without significant lag phase. For unadapted cultures (inocula from simple substrate-glucose to complex substrate-HSW), hydrogen was produced only in the HSW concentration of 1–2 g-VS/L and the lag phase required more than 2 days. After adaptation, hydrogen was produced directly in the HSW feedstock (10 g-VS/L) with the maximum yield of 101.7 ± 9.1 mL H2/g VSadded. Acetic acid was the main fermentation product in all HSW concentration cultivation. Furthermore, hydrogen production was demonstrated in continuous system with adapted cultures while process failure was found with unadapted cultures.
This review summarized several main factors influencing fermentative hydrogen production. The reviewed factors included inoculum, substrate, reactor type, nitrogen, phosphate, metal ion, temperature and pH. In this review, the effect of each factor on fermentative hydrogen production and the advance in the research of the effect were briefly introduced and discussed, followed by some suggestions for the future work of fermentative hydrogen production. This review showed that there usually existed some disagreements on the optimal condition of a given factor for fermentative hydrogen production, thus more researches in this respect are recommended. Furthermore, most of the studies on fermentative hydrogen production were conducted in batch mode using glucose and sucrose as substrate, thus more studies on fermentative hydrogen production in continuous mode using organic wastes as substrate are recommended.
The effect of heat treatment at different temperatures on two types of inocula, activated sludge and anaerobically digested sludge, was investigated in batch cultures. Heat treatments were conducted at 65, 80 and 95 °C for 30 min. The untreated inocula produced less amount of hydrogen than the pretreated inocula, with lactic acid as the main metabolite. The maximum yields of 2.3 and 1.6 mol H2/mol glucose were achieved for the 65 °C pretreated anaerobically digested and activated sludges, respectively. Approximately a 15% decrease in yield was observed with increasing pretreatment temperature from 65 to 95 °C concomitant with an increase in butyrate/acetate ratio from 1.5 to 2.4 for anaerobically digested sludge. The increase of pretreatment temperature of activated sludge to 95 °C suppressed the hydrogen production by lactic acid fermentation. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity.
Direct cellulose fermentation by cellulolytic anaerobic bacteria offers potential to generate renewable hydrogen (H2) from inexpensive “waste” cellulosic feedstocks. The rates and yields of H2 production via direct cellulose fermentation are low and must be increased significantly if this technology is to become a viable method for generating usable H2. A much more comprehensive understanding of the relationships between gene and gene product expression, end-product synthesis patterns, and the factors that regulate carbon and electron balance, within the context of the bioreactor conditions must be achieved if we are to improve molar yields of H2 during cellulose fermentation. Strategies to increase yields of H2 production from cellulose include manipulation of carbon and electron flow via end-product inhibition (metabolic shift), metabolic engineering at the genetic level, synergistic co-cultures, and bioprocess engineering and bioreactor designs that maintain a neutral pH during fermentation and ensure rapid removal of H2 and CO2 from the aqueous phase.
A functional hydrogen producing consortium was isolated from soil by heat pre-treatment technique and hydrogen production at different substrate concentration was evaluated. The forest soil was heat pre-treated at 65, 80, 95, 105 and 120 °C temperature for 1 h. As revealed by PCR-DGGE analysis and hydrogen yield, the hydrogen producing microbial community changed with increase in heat pre-treatment temperatures giving potential hydrogen producing consortium at 95–105 °C soil pre-treatment. The maximum hydrogen production rate, hydrogen yield and cumulative hydrogen with 15–20 g glucose were 1390–1576 mL/L/day, 1.83–1.93 mol H2/mol glucose, and 2966–3146 mL H2/L, respectively. The metabolic pathways shifted from ethanol-type to acetate–formate type as soil pre-treatment temperature increased from 65 to 120 °C. The soil heat pre-treatment approach is effective for isolating hydrogen producing natural Clostridium consortium from the soil as enumerations of the functional strains need specific temperature range to florish.
Hydrogen (H2) and end-product synthesis by Clostridium thermocellum were investigated in batch cultures using cellulosic sources (α-cellulose, shredded filter paper, and delignified wood fibers (DLWs)) and cellobiose at low (), medium (), and high () added substrate concentrations. Cellulosic substrates produced higher total amounts of H2 in high substrate concentration cultures, but better H2 yields at both low and medium substrate concentrations. DLW was shown to be an effective substrate providing an average yield of 1.6 mol glucose. Acetate, ethanol, lactate, and formate were the primary fermentation end products. Acetate yields per mole hexose were highest in low substrate concentration cultures and yields shifted toward increased lactate at high substrate concentrations. On average, the ratio of acetate to ethanol (4:3) stayed roughly constant under all growth conditions tested, while lactate, which was a minor product at the end of fermentation under low and medium sugar concentrations, represented >30% of the organic end products at the end of the fermentation in the presence of high levels of substrate. Since these were unstirred cultures, development of H2 supersaturation may help explain this shift. At low and medium substrate concentrations, H2 production and yields were similar or greater in cultures containing cellulosic substrates compared with cellobiose. Overall, delignified wood was found to be the best candidate for H2 production.
Hydrogen may be produced by a number of processes, including electrolysis of water, thermocatalytic reformation of hydrogen-rich organic compounds, and biological processes. Currently, hydrogen is produced, almost exclusively, by electrolysis of water or by steam reformation of methane. Biological production of hydrogen (Biohydrogen) technologies provide a wide range of approaches to generate hydrogen, including direct biophotolysis, indirect biophotolysis, photo-fermentations, and dark-fermentation. The practical application of these technologies to every day energy problems, however, is unclear. In this paper, hydrogen production rates of various biohydrogen systems are compared by first standardizing the units of hydrogen production and then by calculating the size of biohydrogen systems that would be required to power proton exchange membrane (PEM) fuel cells of various sizes.
The capability of natural anaerobic microflora to produce hydrogen was examined with artificial wastewater containing cellulose. The microflora in sludge compost was found to produce a significant amount of hydrogen (2.4 mol/mol-hexose). Among the fermentation products other than hydrogen and carbon dioxide, the lower fatty acids, mainly acetate and butyrate, constituted more than approximately 90% of the total soluble metabolites.
Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H2/mol sugar and specific hydrogen production rate of 225 mmol of H2/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.
Two novel anaerobic, moderately thermophilic and cellulose-/cellobiose-digesting bacteria, EBR45(T) and EBR596(T), were isolated from anaerobic sludge of a cellulose-degrading methanogenic bioreactor. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belonged to cluster III within the low-G+C-content Gram-positive bacteria. The close relatives of EBR45(T) were Clostridium straminisolvens DSM 16021(T) (sequence identity, 94.6 %) and Clostridium thermocellum DSM 1237(T) (93.4 %). The closest relative of EBR596(T) was Clostridium stercorarium DSM 8532(T) (95.9 %). Both isolates were rod-shaped sporulators, growing optimally at 60 degrees C. EBR45(T) was Gram-staining-reaction-variable and non-motile, formed bright-yellow colonies on solid media, and grew on a relatively narrow range of carbohydrates including cellulose and cellobiose. EBR596(T) was Gram-staining-reaction-negative and motile, formed glossy white colonies and grew on cellobiose and various carbohydrates except cellulose. Major fatty acid compositions were 16 : 0 iso, 16 : 0 and 16 : 0 dimethylacetal (strain EBR45(T)) and 15 : 0 iso, 16 : 0 iso, 15 : 0 anteiso and 17 : 0 anteiso (strain EBR596(T)). The DNA G+C contents were 36.9 mol% (EBR45(T)) and 51.1 mol% (EBR596(T)). Based on the phenotypic and phylogenetic data and genomic distinctiveness, strains EBR45(T) and EBR596(T) represent two novel species, for which the names Clostridium clariflavum sp. nov. (type strain EBR45(T) =DSM 19732(T) =NBRC 101661(T)) and Clostridium caenicola sp. nov. (type strain EBR596(T) =DSM 19027(T) =NBRC 102590(T)) are proposed.
No comprehensive review on the bioconversion of lignocellulosic biomass to hydrogen is presented. This paper provides an up-to-date review on recent research development in biotechnology-based lignocellulosic biomass-to-H(2) conversion. Bioconversion of lignocellulosic prehydrolysate, hydrolysate or cellulose to hydrogen was discussed in terms of the involved microorganisms and the bioaugmentation tactics. To achieve fully the utilization of biomass, the integrated approaches composed of coupled dark-photo fermentation and the dark fermentation and bioelectrohydrogenesis were sketched. Additionally, this review sheds light on the perspectives on the lignocellulosic biomass conversion to hydrogen, and on the scientific and technical challenges faced for the lignocelluloses bioconversion.
A significant effort is underway to develop biofuels as replacements for non-renewable fossil fuels. Among the various options, hydrogen is an attractive future energy carrier due to its potentially higher efficiency of conversion to usable power, low generation of pollutants and high energy density. There are a variety of technologies for biological hydrogen production; here, we concentrate on fermentative hydrogen production and highlight some recently applied approaches, such as response surface methodology, different reactor configurations and organisms that have been used to maximize hydrogen production rates and yields. However, there are significant remaining barriers to practical application, such as low yields and production rates, and we discuss several methods, including two stage processes and metabolic engineering, that are aimed at overcoming these barriers.
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.
A high performance liquid chromatography (HPLC) method was developed for the determination of seven short-chain fatty acids in equine caecal liquor. Samples were cleaned up on a Sep-pak (C18) cartridge, and the analyte was eluted from the extraction cartridge and filtered through a 0.45 micron cellulose nitrate filter. The analyte was chromatographed by ion exchange HPLC. Detection was by UV at 210 nm. Recovery from phosphate buffer (0.05 M, pH 7.0) and equine caecal liquor was 76.95% (lactic), 76.76% (valeric). The limit of (propionic), 89.35% (isobutyric), 88.73% (butyric), 80.33% (isovaleric) and 72.61% (valeric). The limit of detection of the short-chain fatty acids in phosphate buffer was 0.00006 M (lactic), 0.0001 M (acetic), 0.0002 M (propionic), 0.0001 M (isobutyric), 0.0002 M (butyric), 0.0002 M (isovaleric) and 0.0003 M (valeric). The specificity and sensitivity of this method was sufficiently high to allow the characterization of the pattern of these short-chain fatty acids in equine caecal liquor following intravenous administration of oxytetracycline at the recommended dose rate in a pony.
Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized. Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells. The optimum growth temperature is 55 to 60 degrees C, with a range of 40 to 65 degrees C. The deoxyribonucleic acid composition is 35 mol% G + C. The name Clostridium cellulosi sp. nov. is proposed. The type strain is AS 1.1777. Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells. The deoxyribonucleic acid composition is 34 mol% G + C. The name Clostridium cellulofermentans sp. nov. is proposed. The type strain is AS 1.1775. Both C. cellulosi AS 1.1777 and C. cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People's Republic of China.
The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland. The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy. Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose. Fermentation end products were ethanol, acetate, lactate, CO2, and H2. The temperature optimum for growth was between 70 and 75 degrees C, and growth occurred in the range of 50-75 degrees C. The pH range for growth was 4.7-8.8, with an optimum at pH 7.0. Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria. Addition of 50.66 kPa H2 or 2% NaCl did not affect growth. The isolate grew in the presence of exogenously added 4% (w/v) ethanol. The G+C ratio was 37 mol%. 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter. Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed.
A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h−1 with pectin and 0.12 h−1 with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K
m values of both lyases for pectate and citrus pectin were 0.5 g·l−1 and 5.0 g·l−1, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca2+. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov.
Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.
The biological production of hydrogen from the fermentation of different substrates was examined in batch tests using heat-shocked mixed cultures with two techniques: an intermittent pressure release method (Owen method) and a continuous gas release method using a bubble measurement device (respirometric method). Under otherwise identical conditions, the respirometric method resulted in the production of 43% more hydrogen gas from glucose than the Owen method. The lower conversion of glucose to hydrogen using the Owen protocol may have been produced by repression of hydrogenase activity from high partial pressures in the gastight bottles, but this could not be proven using a thermodynamic/rate inhibition analysis. In the respirometric method, total pressure in the headspace never exceeded ambient pressure, and hydrogen typically composed as much as 62% of the headspace gas. High conversion efficiencies were consistently obtained with heat-shocked soils taken at different times and those stored for up to a month. Hydrogen gas composition was consistently in the range of 60-64% for glucose-grown cultures during logarithmic growth but declined in stationary cultures. Overall, hydrogen conversion efficiencies for glucose cultures were 23% based on the assumption of a maximum of 4 mol of hydrogen/ mol of glucose. Hydrogen conversion efficiencies were similar for sucrose (23%) and somewhat lower for molasses (15%) but were much lower for lactate (0.50%) and cellulose (0.075%).
The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.
We examined hydrogen production from a dairy cow waste slurry (13.4 g of volatile solids per liter) by batch cultures in a temperature range from 37 to 85 degrees C, using microflora naturally present within the slurry. Without the addition of seed bacteria, hydrogen was produced by simply incubating the slurry, using the microflora within the slurry. Interestingly, two peaks of fermentation temperatures for hydrogen production from the slurry were observed at 60 and 75 degrees C (392 and 248 ml H2 per liter of slurry, respectively). After the termination of the hydrogen evolution, the microflora cultured at 60 degrees C displayed hydrogen-consuming activity, but hydrogen-consuming activity of the microflora cultured at 75 degrees C was not detected, at least for 24 days. At both 60 and 75 degrees C, the main by-product was acetate, and the optimum pH of the slurry for hydrogen production was around neutral. Bacteria related to hydrogen-producing moderate and extreme thermophiles, Clostridium thermocellum and Caldanaerobacter subterraneus, were detected in the slurries cultured at 60 and 75 degrees C, respectively, by denaturing gradient gel electrophoresis analyses, using the V3 region of 16S rDNA.
Microbial community composition dynamics was studied during H(2) fermentation from glucose in a fluidized-bed bioreactor (FBR) aiming at obtaining insight into the H(2) fermentation microbiology and factors resulting in the instability of biofilm processes. FBR H(2) production performance was characterised by an instable pattern of prompt onset of H(2) production followed by rapid decrease. Gradual enrichment of organisms increased the diversity of FBR attached and suspended-growth phase bacterial communities during the operation. FBR bacteria included potential H(2) producers, H(2) consumers and neither H(2) producers nor consumers, and those distantly related to any known organisms. The prompt onset of H(2) production was due to rapid growth of Clostridium butyricum (99-100%) affiliated strains after starting continuous feed. The proportion trend of C. butyricum in FBR attached and suspended-growth phase communities coincided with H(2) and butyrate production. High glucose loading rate favoured the H(2) production by Escherichia coli (100%) affiliated strain. Decrease in H(2) production, associated with a shift from acetate-butyrate to acetate-propionate production, was due to changes in FBR attached and suspended-growth phase bacterial community compositions. During the shift, organisms, including potential propionate producers, were enriched in the communities while the proportion trend of C. butyricum decreased. We suggest that the instability of H(2) fermentation in biofilm reactors is due to enrichment and efficient adhesion of H(2) consumers on the carrier and, therefore, biofilm reactors may not favour mesophilic H(2) fermentation.
Hydrogen and methane co-production from potato waste was examined using a two-stage process of anaerobic digestion. The hydrogen stage was operated in continuous flow under a pH of 5.5 and a HRT of 6h. The methane stage was operated in both continuous and semi-continuous flows under HRTs of 30 h and 90 h, respectively, with pH controlled at 7. A maximum gas production rate of 270 ml/h and an average of 119 ml/h were obtained from the hydrogen stage during the operation over 110 days. The hydrogen concentration contained in the gas was 45% (v/v), on average. The maximum and average gas production rates observed from methane reactor during the 74 days of semi-continuous flow operation were 187 and 141 ml/h, respectively, with an average methane concentration of 76%. Overall, 70% of VS, 64% of total COD in the feedstock were removed. The hydrogen and methane yields from the potato waste were 30 l/kg TS (with a maximum of 68 l/kg) and 183 l/kg TS (with a maximum of 225 l/kg), respectively. The total energy yield obtained was 2.14 kW h/kg TS, with a maximum of 2.74 kW h/kg TS.
Microbial fermentations are potential producers of sustainable energy carriers. In this study, ethanol and hydrogen production was studied by two thermophilic bacteria (strain AK15 and AK17) isolated from geothermal springs in Iceland. Strain AK15 was affiliated with Clostridium uzonii (98.8%), while AK17 was affiliated with Thermoanaerobacterium aciditolerans (99.2%) based on the 16S rRNA gene sequence analysis. Both strains fermented a wide variety of sugar residues typically found in lignocellulosic materials, and some polysaccharides. In the batch cultivations, strain AK17 produced ethanol from glucose and xylose fermentations of up to 1.6 mol-EtOH/mol-glucose (80% of the theoretical maximum) and 1.1 mol-EtOH/mol-xylose (66%), respectively. The hydrogen yields by AK17 were up to 1.2 mol-H2/ mol-glucose (30% of the theoretical maximum) and 1.0 mol-H2/mol-xylose (30%). The strain AK15 produced hydrogen as the main fermentation product from glucose (up to 1.9 mol-H2/mol-glucose [48%]) and xylose (1.1 mol-H2/mol-xylose [33%]). The strain AK17 tolerated exogenously added ethanol up to 4% (v/v). The ethanol and hydrogen production performance from glucose by a co-culture of the strains AK15 and AK17 was studied in a continuous-flow bioreactor at 60 degrees C. Stable and continuous ethanol and hydrogen co-production was achieved with ethanol yield of 1.35 mol-EtOH/mol-glucose, and with the hydrogen production rate of 6.1 mmol/h/L (H2 yield of 0.80 mol-H2/mol-glucose). PCR-DGGE analysis revealed that the AK17 became the dominant bacterium in the bioreactor. In conclusion, strain AK17 is a promising strain for the co-production of ethanol and hydrogen with a wide substrate utilization spectrum, relatively high ethanol tolerance, and ethanol yields among the highest reported for thermoanaerobes.
Biological hydrogen production measured in batch anaerobic respirometers
- Logan
Effect of fermentation temperature on hydrogen production from cow waste slurry by using anaerobic microflora within the slurry
- Yokoyama