No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Microbial diversity of hydrogen-producing bacteria in batch reactors fed with cellulose using leachate as inoculum
Abstract
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
... Lactic acid was determined by liquid chromatography (HPLC, Shimadzu®), using ultraviolet detector with SCL-M10AVP diode array and Aminex® HPX-87H (300 mm × 7.8 mm; Bio-Rad). The H 2 biogas content was monitored by gas chromatography (GC 2010, Shimadzu®) through a Carboxen™ 1010 PLOT capillary column (30 m × 0.53 mm, Supelco) and argon as the carrier gas [22]. ...
... Representative of this genus is characterized by the presence of cellulosome, which contains cellulolytic and hemicellulolytic enzymes for the production of butyric acid and H 2 with high yield [43]. This result corresponds to that observed by Ratti et al. (2013) in batch reactors fed with cellulose, pH 7.0 and 37 °C [22]. The authors found a relative abundance greater than 98% for Clostridium sp. and maximum H 2 production of 51.52 mLH 2 /mol cellulose and butyric acid (1493 mg/L), with cellulose degradation of 44.7%. ...
... Representative of this genus is characterized by the presence of cellulosome, which contains cellulolytic and hemicellulolytic enzymes for the production of butyric acid and H 2 with high yield [43]. This result corresponds to that observed by Ratti et al. (2013) in batch reactors fed with cellulose, pH 7.0 and 37 °C [22]. The authors found a relative abundance greater than 98% for Clostridium sp. and maximum H 2 production of 51.52 mLH 2 /mol cellulose and butyric acid (1493 mg/L), with cellulose degradation of 44.7%. ...
In this research batch reactors were operated with coffee processing waste and autochthonous microbial consortium, and a taxonomic and functional analysis was performed for phase I of stabilization of maximum H2 production and for phase II of maximum H2 consumption. During phase I, the reactor’s operating conditions were pH 4.84 to 8.18, headspace 33.18% to 66.82%, and pulp and husk from 6.95 to 17.05 g/L. These assays continued for phase II, with initial pH conditions of 5.8–8.1, headspace of 33.18–66.82%, and pulp and husk remaining from phase I. The highest homoacetogenesis was observed in assay 5 with pH 7.7, 40% headspace, and 15 g/L of pulp and husk (initial concentrations of phase I). A relative abundance of Clostridium 41%, Lactobacillus 20% and Acetobacter 14% was observed in phase I. In phase II, there was a change in relative abundance of 21%, 63%, and 1%, respectively, and functional genes involved with autotrophic (formyltetrahydrofolate synthase) and heterotrophic (enolase) homoacetogenesis, butanol (3-hydroxybutyryl-CoA dehydrogenase), and propionic acid (propionate CoA-transferase) were identified. This study provides a new and amplified insight into the physicochemical and microbiological factors, which can be used to propose adequate operational conditions to maximize the bioenergy production and reduce homoacetogenesis in biological reactors.
Graphical abstract
... Microbial consortia have a higher threshold of dealing with complex substrates due to high microbial diversity, which reduces handling and operational cost considerably [18,19]. Inocula from different anaerobic environments (leachate, elephant dung, SCB, upflow anaerobic sludge blanket reactors) have been used for hydrogen production [15,16,20,21]. Nevertheless, mixed cultures can bring disadvantages since they certainly contain methanogenic archaea and homoacetogens which promotes the consumption of hydrogen [22]. ...
... The most common pretreatments employed to inhibit methanogenic archaea in anaerobic inoculum are heat, alkaline, acidification, ultrasonic and heat shock pretreatments [21e24]. The acid and heat shock pretreatment of the inoculum (rumen fluid and leachate) inhibited the cellulolytic bacteria, since H 2 was produced from cellulosic residues only in the presence of the cellulase enzyme [21,23]. In order to apply a treatment to the inoculum that did not affect the fermentative and cellulolytic bacteria a different method was applied in this study. ...
... For the hydrolysis of cellulose, Ratti et al. [21,24] employed commercial cellulase enzymes and as the inoculum source the authors used pretreated and leached rumen fluid. The authors concluded that the inoculum pretreatment (acid and heat shock) may have inhibited the cellulolytic organisms, since H 2 was produced only in the presence of the cellulase enzyme. ...
The influence of 2-bromoethanesulfonate (BES) on anaerobic inocula, from a Wastewater Treatment Plant, was evaluated for enriching the hydrogen (H2) production using sugarcane bagasse (SCB). The assays were carried out in 100 mL reactors at 37 °C under five conditions: A (without BES), B (Inoculum pretreated with 20 mM BES – 24 h), C (Inoculum pretreated with 20 mM BES – 30 min), D (10 mM BES in the culture medium) and E (20 mM BES in the culture medium). H2 production reached the highest value (10.96 mmol/L) for condition D. Methane was detected in B (0.43 mmol/L) and C (0.73 mmol/L). The addition of BES in the culture medium inhibited the consumption of H2 by methanogens. H2 was produced primarily through the fermentation of acetic acid. Despite the absence of methane, Archaea was detected in sample D. The inoculum was favorable for H2 production using SCB and the addition of 10 mM BES in the culture medium was the best strategy employed.
... Temperature is an important factor that influences the bioconversion of cellulose to hydrogen. Mesophilic and thermophilic conditions have been commonly used in studies of H 2 production [5,10,11]. Datar et al. [5] and Ratti et al. [11] reported that mesophilic anaerobic bacteria are not capable of utilizing cellulose; it is necessary to add exogenous cellulase. In contrast, thermophilic anaerobic bacteria can effectively hydrolyze the cellulose [10]. ...
... Mesophilic and thermophilic conditions have been commonly used in studies of H 2 production [5,10,11]. Datar et al. [5] and Ratti et al. [11] reported that mesophilic anaerobic bacteria are not capable of utilizing cellulose; it is necessary to add exogenous cellulase. In contrast, thermophilic anaerobic bacteria can effectively hydrolyze the cellulose [10]. ...
... The hydrogen content in the biogas was determined by gas chromatography using a GC 2010 Shimadzu system according to Ratti et al. (2013). The H 2 yield is defined as the amount of H 2 accumulation per amount of the substrate added. ...
H2-production was evaluated using a microbial consortium from sugarcane bagasse in natura (SCBn) to ferment cellulose from pretreated SCB (SCBp; steam explosion and delignification alkaline). Six batch assays were performed: (1) 0.5 g/l SCBn + 0.5 g/l SCBp; (2) 2.0 g/l SCBn + 2.0 g/l SCBp; (3) 0.5 g/l SCBn + 0.5 g/l glucose; (4) 2.0 g/l SCBn + 2.0 g/l glucose; (5) 0.5 g/l SCBn; (6) 2.0 g/l SCBn. H2-production was verified in all assays; however, the SCBp influenced the H2-production, since that was verified a higher production. The higher H2-yield was in assay 1 (1.2 mol H2/g substrate). The metabolites produced were ethanol and acetic, butyric, iso-butyric, lactic and propionic acids. In assays 1, 2, 5 and 6, the H2-production occurred primarily via acetic acid. In assays 3 and 4, the main metabolite was butyric acid. Microorganisms identified were grouped into distinct phyla: Firmicutes, Bacteroidetes and Actinobacteria, with a predominance of genera Clostridium and Tepidimicrobium that produce cellulolytic enzymes favoring the H2-production from SCB.
... H 2 can be produced by pure or mixed cultures from various simple substrates (glucose, sucrose and xylose) and from waste generated by industry (Kapdan and Kargi 2006;Ratti et al. 2013). The H 2 production from carbohydrates is accompanied by formation of organic acids, CO 2 and other intermediates. ...
... The H 2 production from carbohydrates is accompanied by formation of organic acids, CO 2 and other intermediates. During the anaerobic acidification of carbohydrates, methanogenic microorganisms consume H 2 and have a negative impact on H 2 production; therefore, many studies have attempted to inhibit hydrogen consumers by pH control, heat-shock, or aeration (Ratti et al. 2013). ...
... Another important factor that affects H 2 production is temperature. Mesophilic and thermophilic conditions have been commonly used in studies on H 2 production (Datar et al. 2007;Ratti et al. 2013). However, thermophilic fermentation presents a number of advantages in comparison to the mesophilic process, such as higher yields of H 2 , more efficient degradation of organics, and more resistance to contamination (Zhang et al. 2003). ...
In this study, the composition and diversity of the bacterial community in thermophilic H2-producing reactors fed with glucose were investigated using pyrosequencing. The H2-producing experiments in batch were conducted using 0.5 and 2.0gl(-1) glucose at 550 °C. Under the two conditions, the H2 production and yield were 1.3 and 1.6mol H2mol glucose(-1), respectively. Acetic, butyric, iso-butyric, lactic and propionic acids were detected in the two reactors. The increase in substrate concentration favored a high H2 yield. In this reactor, a predominance of acetic and iso-butyric acids, 27.7% and 40%, were measured, respectively. By means of pyrosequencing, a total of 323 and 247 operational taxonomic units were obtained, with a predominance of the phylum Firmicutes (68.73-67.61%) for reactors with 0.5 and 2.0gl(-1) glucose, respectively. Approximately 40.55% and 62.34% of sequences were affiliated with Thermoanaerobacterium and Thermohydrogenium, microorganisms that produce H2 under thermophilic conditions.
Copyright © 2015 Elsevier GmbH. All rights reserved.
... This process directly uses the crude fermented product as an enzyme source, reducing the production cost in terms of low energy requirement and high product yield [21]. Ratti et al. also investigated the H 2 production from cellulose using rumen fluid as the inoculum and gave the maximum production of 18.5 mmol H 2 [16]. They found that H 2 was produced from cellulose primarily through the fermentation of butyric acid, a route typical of Clostridium species. ...
... Meanwhile, Ratti et al. reported the same bacteria, which also produces acetic and butyric acid and gave 19.9 mmol H 2 . In order to promote the growth of those bacteria, they have added biotin and p-aminobenzoic acid and gave the H 2 yield of 2.30 mol H 2 /mol glucose [16]. This phenomenon shows C. acetobutylicum required the assisted chemical for growth development and formation of cellobiase and cellulase and, thus, needed a longer time for those processes. ...
Biohydrogen produced from cellulosic feedstock is a promising candidate for future energy needs as a renewable energy carrier. The thermochemical route and biological processes have great potential for biohydrogen production. In particular, pyrolysis/gasification and dark fermentation are the methods to enhance the biohydrogen production from cellulose. The review compiles the essential information on both processes, including pretreatment of cellulose since it has a complex structure. The operating conditions for both processes, for example, the influence of cellulose pyrolysis/gasification such as temperature, heating rate, and vapor residence time, while for dark fermentation, including the temperature, inoculum source, hydraulic retention time, and pH, are discussed. The bioreactor configurations and economic aspects of both processes are also discussed. The review aims are to present the current state of knowledge about the two processes using cellulose as substrates. Surprisingly, dark fermentation is a promising method for application of cellulose for biohydrogen production since many works were done on dark fermentation compared to pyrolysis/gasification. The future perspectives on enhancing hydrogen production from cellulose have also been discussed.
... The quantification of H 2 gas was performed by gas chromatography (GC 2010, Shimadzu ® ) through Carboxen™ 1010 PLOT (30 m  0.53 mm, Supelco) capillary column with argon as carrier gas [30]. Soluble metabolites (organic acids and alcohols) were analysed by gas chromatography (GC 2010, Shimadzu ® ), using HP-INNOWAX column (30 m  0.25 mm x 0.25 mm) with flame ionization detector (FID), hydrogen as carrier gas, synthetic air and nitrogen as auxiliary gases, and automatic injection (COMBI-PAL sampler AOC5000, Shimadzu ® ) [30]. ...
... The quantification of H 2 gas was performed by gas chromatography (GC 2010, Shimadzu ® ) through Carboxen™ 1010 PLOT (30 m  0.53 mm, Supelco) capillary column with argon as carrier gas [30]. Soluble metabolites (organic acids and alcohols) were analysed by gas chromatography (GC 2010, Shimadzu ® ), using HP-INNOWAX column (30 m  0.25 mm x 0.25 mm) with flame ionization detector (FID), hydrogen as carrier gas, synthetic air and nitrogen as auxiliary gases, and automatic injection (COMBI-PAL sampler AOC5000, Shimadzu ® ) [30]. ...
The objective of this study was to screen the factors that affect H2, organic acids and alcohols production from coffee waste pretreated in a hydrothermal reactor applying consortium of bacteria and fungi (indigenous from coffee waste) with hydrolytic and fermentative activity. The effects of pH (4.0–7.0), temperature (30–50 °C), agitation (0–180 rpm), headspace (50–70%), percentage of bioaugmentation (without microbial consortium to 20%), concentration of coffee pulp and husk (2–6 g/L), coffee processing wastewater (7-30 gCOD/L) and yeast extract (0–2 g/L) were evaluated using a Plackett-Burman design. The highest H2 production potential (82 ml H2) was obtained under the following conditions: 30 °C, 180 rpm, 50% headspace, without bioaugmentation, 2 g/L pulp and husk coffee, 30 gCOD/L coffee processing wastewater and 2 g/L yeast extract. The main soluble products were acetic acid (1956 mg/L), lactic acid (786 mg/L) and ethanol (816 mg/L). Lactobacillus sp., Clostridium sp., Saccharomyces sp. and Kazachstania sp. were the main autochthonous microorganisms identified. Through metagenome functional analysis, enzymes related to lignin, phenol, cellulose, lignocellulose, and pectin degradation were identified, as well as acidogenesis, and H2 production.
... There are several types of microbial consortium, in which members have complementary metabolic activities, performing complex function that a single microorganism cannot achieve, such as cow dung compost [19], rumen fluid [22] leachate samples [23] and thermophilic sludge [24]. ...
... The gases production (H 2 , CO 2 and CH 4 ) was monitored by gaseous chromatography (GC) 2010 (Shimadzu, Japan) equipped with a thermal conductivity detector (TCD) and a Carboxen 1010 PLOT column (30 m × 0.53 mm) [23]. Argon gas was used as the carrier gas at 12 mL min −1 flow rate, with The determination of volatile organic acids, reducing sugars (such as maltose) and alcohols was carried out by High Performance Liquid Chromatography (HPLC Shimadzu system), equipped with a UV diode array detector (SDP-M10Avp), a refraction index detector (RID-10A), a CTO-20A oven, a LC-10AD vp Pump and a SCL 10 A vp controller, with self-injector (SIL-20-Avp). ...
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.
... Previous studies have tried to identify the microorganisms involved in the different phases of AD to assess the impact they have on the production of methane. Species belonging to the genus Clostridium are active during the hydrolysis of polysaccharides and proteins in the AD process (Burrell et al., 2004;Cirne et al., 2007;Doi, 2008;Ratti et al., 2013;Schnü;rer and Jarvis, 2010). Several Clostridium spp. ...
Pathogenic spore-forming Firmicutes are commonly present in animal and human wastes that are used as fertilizers in crop production. Pre-treatments of organic waste prior to land application offer the potential to abate enteric microorganisms, and therefore reduce the risk of contamination of crops or adjacent water resources with pathogens carried in these materials. The inactivation and reduction of gram-positive spore formers such as Clostridium spp., Clostridioides spp. and Bacillus spp. from animal and human waste can be challenging given the recalcitrance of the spores these bacteria produce. Given the significance of these organisms to human and animal health, information concerning spore-forming bacteria inactivation during anaerobic digestion (AD) and aerobic composting (AC) is required as the basis for recommending safe organic waste management practices. In this review, an assessment of the inactivation of spore-forming Firmicutes during AD and AC was conducted to provide guidance for practical management of organic matrices of animal or human origin. Temperature and pH may be the main factors contributing to the inactivation of spore-forming Firmicutes during batch lab-scale AD (log reduction <0.5–5 log). In continuous digesters, wet AD systems do not effectively inactivate spore-forming Firmicutes even under thermopholic conditions (log reduction −1.09 – 0.98), but dry AD systems could be a feasible management practice to inactivate spore-forming Firmicutes from organic materials with high solid content (log reduction 1.77–3.1). In contrast, composting is an effective treatment to abate spore-forming Firmicutes (log reduction 1.7–6.5) when thermophilic conditions last at least six consecutive days. Temperature, moisture content and composting scale are the key operating conditions influencing the inactivation of spore-forming Firmicutes during composting. Where possible, undertaking AD with subsequent composting to ensure the biosafety of digestate before its downstream processing and recycling is recommended to abate recalcitrant bacteria in digestate.
... The cost-effective pretreatment of waste and wastewater containing cellulose is a challenge to overcome the socio-economic factors [13][14][15][16][17]. Actually, some species the genera Clostridium and Enterobacter have been widely described as cellulosic degrading microflora [18][19][20][21]. These bacteria are found in different anaerobic environments such as digest sludge, rumen, landfill leachate, and soil [21][22][23][24]. However, in our previous studies, the non-cellulolytic bacteria contribute to cellulose bioenergy recovery by controlling pH and consuming metabolites, factors which influence the cellulolytic activity by enzyme inhibition and metabolite repression [24]. ...
The efficiency of single-stage biohythane production from synthetic cellulosic wastewater under mesophilic temperature was investigated. A lab-scale continuously stirred tank reactor was conducted at a hydraulic retention time of 10 days using mixed microflora without pretreatment. The experimental results show that a stable methane and hydrogen yields of 18.2 ± 0.16 and 5.6 ± 0.31 L/kg VS were maintained for 240 days, respectively with acetate/butyrate ratio of 0.39 as the main byproducts. Based on COD mass balance, more than 45% of the decomposed COD converted to bio-hythane, which means that the setting temperature 37 °C and pH improved the conditions of degradation efficiency. The energy recovery calculations indicated that the total net energy was 4.54 MJ/m³ of cellulosic wastewater. This work contributes to the limited knowledge on continuous cellulosic-hythane conversion into a safe and clean form of energy.
Graphic Abstract
Open image in new window
... The mobile phase consisted of H 2 SO 4 (0.01 N) at 0.5 mL min −1 flow rate [25]. The hydrogen and methane contents in the biogas were determined by gas chromatography using a GC 2010 Shimadzu system equipped with a thermal conductivity detector (TCD) and a Carboxen 1010 PLOT column (30 m × 0.53 mm) according to Ratti and co-workers [26]. ...
The genera Dysgonomonas, Coprococcus, Sporomusa, Bacteroides, Sedimentibacter, Pseudomonas, Ruminococcus, and Clostridium predominate in compost residue, and vadimCA02, Anaerobaculum, Tatlockia, Caloramator, and Syntrophus prevail in soil used as inoculum in batch rectors. This mixed consortium was used as inoculum for biogas production using different concentrations of sugarcane bagasse (SCB) (from 1.58 to 4.42 g/L) and yeast extract (YE) (from 0.58 to 3.42 g/L) according to a composite central design. The maximum ethanol production (20.11 mg L⁻¹) was observed using 2.0 and 3.0 g L⁻¹ of YE and SCB, respectively (C6). Likewise, the highest hydrogen production (0.60 mmol L⁻¹) was observed using 3.0 and 4.0 g L⁻¹ of YE and SCB, respectively (C1). Methane was also observed, reaching the maximum production (1.44 mmol L⁻¹) using 1.0 and 4.0 g L⁻¹ of YE and SCB, respectively (C2). The archaeal similarity between these conditions was above 90%; however, the richness and diversity were higher in the C2 (12 and 2.42, respectively) than in C1 (5 and 1.43, respectively) and C6 (11 and 2.29, respectively). Equally, the bacterial similarity between C1 and C6 was 60% while richness of 24 and 17 and diversity of 3.13 and 2.81 were observed in C1 and C6, respectively.
... Inhibitory effects vary among bacteria, however. It is plausible that cellulose biodegradation at saturation enhances organic acid production, which may decrease the pH to prohibitively low levels [73]. Continuous culture, fed-batch or plug flow systems rather than batch cultures at a range of cellulose concentrations should give further insight into saturation effects. ...
In this study, we hypothesized that anaerobic biodegradation of cellulose is influenced by cellulose type and concentration, temperature, and their interactions. Cellulose biodegradation by an anaerobic consortium was tested in thermophilic batch experiments that combined cellulase action, hydrolysis, and fermentation. Initially, the main constituents in the inocula were Thermoanaerobacter, Clostridium, and Acetivibrio spp. Four types of cellulose and a range of concentrations were used as feedstock with pathways involving hydrolysis and glycolysis to produce H 2 , CO 2 , acetate, and ethanol. Long fibrous cellulose, two types of microcrystalline cellulose, and filter paper squares were tested at several concentrations between 2 and 20 g/l as substrates. The yields ranged between 0.1 and 2.9 mmol H 2 and 0.7–2.6 mmol ethanol per g cellulose. The rates ranged between 0.01 and 0.2 mmol H 2 , 0.03–0.2 mmol CO 2 , and 0.01–0.05 mmol ethanol per g cellulose·h. Statistical analyses indicated that the rates and yields of metabolite production were influenced by two-way interactions between the temperature, type, and concentration of cellulose. The results suggest that two-way interactions between experimental variables may impact the outcomes in cellulose bioconversion studies.
... The hydrogen and methane were determined by gas chromatography using a GC 2010 Shimadzu system equipped with a thermal conductivity detector (TCD) and a Carboxen 1010 PLOT column (30 m × 0.53 mm) according to [13]. The organic acids and ethanol were analysed by high-performance liquid chromatography (HPLC) equipped with a UV diode array detector (SPD-M10 AVP), a refraction index detector (RID-10 A), a CTO-20 A oven an LC-10 ADVP Pump, an SCL 10 AVP control and column HPX-87H from Aminex, with 300 mm × 7.8 mm (BioRad). ...
The substrate and yeast extract (YE) concentrations of a batch reactor were changed according to a central composite design in order to optimize the conversion of sugarcane bagasse (SCB) into hydrogen. The optimum hydrogen production (1.50 mmol/L) was obtained using 2.77 and 5.84 g/L of YE and SCB, respectively. Taxonomic analysis using the SEED database indicated that Clostridium (33% of total communities) and Methanothermobacter (40% of archaeal community) were the most abundant genera in the high hydrogen performance reactor. Key microorganisms and related pathways involved in all steps of the anaerobic digestion were further revealed and may help drive sugarcane bagasse bioconversion.
... Phummala et al. (2014) found a combination of physical, chemical and biological treatment method can significantly enhance the hydrolysis of biomass. Ratti et al. (2013) enhanced hydrogen production and substrate degradation through the addition of specific enzymes into fermentation system, and hydrolysis was promoted during the fermentation process. Considering the low substrate degradation rate in all test groups, addition of enzymes are recommended to further promote the hydrolysis of algae biomass during the fermentation process, and thus enhancing the substrate degradation rate and hydrogen production simultaneously. ...
Suitable pretreatment can effectively enhance the fermentative hydrogen production from algae biomass. In this study, combined microwave-acid pretreatment was applied to disintegrate the biomass of macroalgae L. japonica, and dark fermentation in batch mode was conducted for hydrogen production. The results showed that combining microwave pretreatment at 140 °C and 2450 MHz with 1% H2SO4 for 15 min could effectively disrupt macroalgal cells and release the organic matters, and soluble chemical oxygen demand (SCOD) concentration increased by 1.92-fold and achieved 5.12 g/L. During the fermentation process, both polysaccharides and proteins were consumed. Hydrogen production process was dominated by acetate-type fermentation, and the dominance of genus Clostridium contributed to more efficient hydrogen production. After the pretreatment, hydrogen yield increased from 15 mL/g TSadded to 28 mL/g TSadded, and energy conversion efficiency increased from 9.5% to 23.8%. Combined microwave-acid pretreatment is potential in enhancing hydrogen production from the biomass of L. japonica.
... The experimental data were fitted to the mean values of the triplicate sets of reactors using the Statistica 7.0. The average of the hydrogen evolution data was adjusted to the modified Gompertz model (Zwietering et al., 1990), which has been described as a suitable model for the adjustment of accumulated biogas production data in batch experiments (Ratti et al., 2014(Ratti et al., , 2013. ...
... Previous studies showed that Bacteriodietes played an important role in nitrogen removal system ( Meesap et al., 2012). In addition, phylum Firmicutes mainly exited in anoxic zone rather than in oxic zone; class Clostridia was commonly found in cellulolytic environment ( Goberna et al., 2009), which was able to degrade the cellulose in landfill leachate ( Ratti et al., 2013); and Planctomycetes (mainly Phycisphaerae) existed largely in MBR. There were also some bacteria with a small proportion, for example, Deinococcus-Thermus accounted for 2.32-4.39%, ...
... The pH alteration can inhibit the hydrogenase enzyme and change the metabolic pathway to solventogenesis, hence decreasing the H 2 production [47]. Fangkum and Reungsang [48] observed maximum H 2 production at pH 6.5 also using SCB as substrate and elephant dung as inoculum. ...
Hydrogen (H2) production by Clostridium cellulolyticum was investigated. Anaerobic batch reactors were operated with cellobiose (2 g/L) and pretreated sugarcane bagasse (SCB) (2 g/L) using a hydrothermal system to observe the effects of carbon source on H2 production. Salts (NH4Cl, NaCl, MgCl2 and CaCl2) and vitamins (biotin, nicotinamide, p-aminobenzoic acid, thiamine, pantothenic acid, pyridoxamine, cyanocobalamin, riboflavin, folic and lipoic acid) were supplemented from stock solutions at different volumes percentages, ranging from 0 to 5%. The optimal concentration was 2.5% and the strain used both substrates and produced H2 which was higher for cellobiose (14.9 ± 0.2 mmol/L) than for SCB (7.6 ± 0.2 mmol/L), although the λ phase was much smaller when SCB (59.9 h) was used in relation to the assay with cellobiose (164 h). H2 was produced from SCB primarily through the fermentation of lactic and acetic acids.
... Landfill leachate has also been considered a low-cost substrate for fermentative H 2 production [9,10]. Studies on H 2 production have included landfill leachate as inoculum [11,12] and nutrient source [13]. Landfill leachate originates from sanitary landfill and contains a great amount of poorly biodegradable organic material and toxic metals, which makes it highly polluting [14]. ...
Fermentation can use renewable raw materials as substrate, which makes it a sustainable method to obtain H2. This study evaluates H2 production by a mixed culture from substrates such as glucose and derivatives from sugarcane processing (sucrose, molasses, and vinasse) combined with landfill leachate. The leachate alone was not a suitable substrate for biohydrogen production. However, leachate blended with glucose, sucrose, molasses, or vinasse increased the H2 production rate by 2.0-, 2.8-, 4.6-, and 0.5-fold, respectively, as compared with the substrates without the leachate. Determination of metals (Cu, Cd, Pb, Hg, Ni, and Fe) at the beginning and at the end of the fermentative assays showed how they were consumed during the fermentation and demonstrated improved H2 production. During fermentation, Cu, Fe, and Cd were the most consumed leachate metals. The best substrate combination to produce H2 was molasses and leachate, which gave high volumetric productivity—469 ml H2/l h. However, addition of the leachate to the substrates stimulated lactic acid formation pathways, which lowered the H2 yield. The use of leachate combined with sugarcane processing derivatives as substrates could add value to the leachate and reduce its polluting power, generating a clean energy source from renewable raw materials.
... Hydrogen yield of 0.6-19 mmol H2/g cellulose was achieved, and higher yield was obtained from thermophilic fermentation systems. (Ratti et al., 2013) As to the natural cellulose-based biomass, various straws were most widely studied for hydrogen production. As shown in Table 4-10, 10-340 g TS /L straw was used as substrate after pretreatment. ...
Various organic wastes can be used as low-cost substrate for fermentative hydrogen production, which significantly reduces the hydrogen production cost. Furthermore, biohydrogen production from organic wastes can achieve dual benefits of clean energy generation and waste management since agricultural and municipal wastes can be disposed at the same time. In this chapter, various organic wastes as feedstock, including waste activated sludge produced from wastewater treatment plant, algae, agricultural residuals and municipal wastes, for biological hydrogen production, was reviewed. Since low hydrolytic enzymatic activity is observed with the anaerobic cultures, a pretreatment step is often required for the hydrolysis of organic wastes to enhance the hydrogen production efficiency. Pretreatment process can destroy the crystal structure of macromolecular substances and reduce their polymerization degree. Therefore, the trapped components can be released through cell wall lysis and delignification of lignocellulosic biomass to make higher proportion of readily fermentable substances accessible for microorganisms. Various pretreatment methods used for treating organic wastes as feedstock for hydrogen production were analyzed and compared. Physical treatment, chemical treatment, biological treatment and a combination of different treatments are usually used. Physical treatment methods include mill, grind, ultrasonication, heat, freeze and thaw, microwave and ionizing radiation; chemical treatment methods comprise acid and alkaline treatment, oxidation by oxidizing agent and addition of methanogenic inhibitors; biological treatment methods mainly consist of enzymatic treatment and bacterial hydrolysis. Pretreatment is a critical process for fermentative hydrogen production from biomass. Considerable efforts are needed from both technical and managing aspects to achieve a full-scale application of fermentative hydrogen production from biomass.
... H 2 is a promising candidate of ideal fuel in the future due to its natures of being clean and with a high energy yield of 122 kJ/g (Ren et al., 2007), which is 2.75 times the energy content of hydrocarbon fuels (Koutrouli et al., 2006). Thus, the research direction for H 2 energy is to explore how to use the energy carrier of H 2 as renewable energy (Lo et al., 2013; Mohammadi et al., 2012; Ratti et al., 2013). The methods of bio-hydrogen production include light-dependent methods (Eroglu and Melis, 2011; Hallenbeck, 2011; Keskin et al., 2011) such as the direct and indirect bio-photolysis method and the photo-fermentation method (Abo-Hashesh et al., 2011), and not light-dependent methods, including the so that H 2 production microflora can be separated efficiently (Kim et al., 2006). ...
This study aimed to explore the influences of single-chamber systems with different applied voltage on bio-hydrogen (H2) production. The reactor used was the bio-electrochemically assisted microbial reactor (BEAMR) membrane-less (BEAMR-membrane-less, BML). The microbial dark fermentative H2 production method was adopted. After the hot screening process and the DNA sequencing, the domesticated dominant microflora was Clostridium sp. This study discussed the influences of the cases with (continuous and intermittent) and without applied voltage separately. The results showed that, the H2 production rate of the case with intermittent applied voltage (117 mL/h g VSS) of 0.24 V was increased of 1.7 folds higher than the without applied voltage (69 mL/h g VSS) and 1.3 folds higher than the case with continuous applied voltage (88.2 mL/h g VSS) of 0.24 V. The produced H2 concentration with intermittent applied voltage was 18.9% (18.6-19.1%) higher than the without applied voltage, while there was no significant difference with continuous applied voltage. © 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Solid and liquid fractions of hydrothermally pretreated sugarcane bagasse (SCB) were simultaneously used as substrate of a novel continuous compartmentalized reactor. The effect of four (56, 42, 28, and 14 hours) hydraulic retention time (HRT) and chemical oxygen demand (COD) (0.5, 3.0, and 9.0 g/L) were evaluated on hydrogen (H 2 ) and organic acids production. Higher H 2 production and yield (686 mL and 1.63 mol/mol consumed carbohydrates, respectively) were obtained at HRT of 28 h, probably due to the Clostridium and Thermoanaerobacterium metabolisms, which accounted for almost 60% of the microbial relative abundance. Under lower and higher HRT (14 and 56 h, respectively) lactic acid predominated without hydrogen production. Other value-added chemical such as citric, valeric and caproic acids were also obtained as a function of HRT. From the functional point of view, enzymes from the glycoside hydrolases group (GHs) potentially performed important role in the lignocellulosic biomass bioconversion.
Screening new Clostridium strains that can efficiently utilize lignocellulose to produce hydrogen is extremely important for dark fermentative hydrogen production. In this study, a mesophilic hydrogen-producing bacterium, identified as Clostridium populeti FZ10, was newly isolated from compost acclimated by microcrystalline cellulose. The strain could produce hydrogen from various cellulosic substrates. The performances of hydrogen production from microcrystalline cellulose (MCC) and corn stalk (CS) were especially investigated. The maximum hydrogen yield and hydrogen production rate from MCC were 177.5 ± 4.8 mL/g and 7.7 ± 0.2 mL·g−1·h−1, respectively. Furthermore, scanning electron microscopy (SEM) images showed that the structure of CS was destroyed after fermentation, which could be attributed to the presence of exoglucanase, endoglucanase, β-glucosidase and xylanase produced by Clostridium populeti FZ10. The maximum hydrogen yield and hydrogen production rate from CS were 92.5 ± 3.7 mL/g and 5.9 ± 0.2 mL·g−1·h−1,respectively, with a cellulose degradation of 47.2 ± 2.3% and a hemicellulose degradation of 58.1 ± 2.0%. This study demonstrates that Clostridium populeti FZ10 is an ideal candidate for directly converting lignocellulose into biohydrogen under mesophilic conditions. The discovery of strain C. populeti FZ10 has special significance in the field of bioenergy.
In order to solve the problem of shortage of carbon source for biological denitrification in advanced treatment of the effluent from secondary treatment of sewage, five kinds of fruit shells (pistachio shell, peanut shell, ginkgo shell, walnut shell and hazelnut shell) were preliminarily selected from eight kinds of fruit shells for experiments of static carbon release and denitrification. The carbon release performance (amount and law of carbon release and biodegradability of released carbon) and denitrification performance of different shells were investigated. Results showed that the peanut shell had the largest amount of carbon release (0.88 mg chemical oxygen demand [COD] g⁻¹) and the highest removal rate of nitrate (NO3⁻-N) (76.48% ± 4.06%). However, the released carbon could not be fully utilized by denitrifying bacteria, resulting in a (205.90% ± 59.49%) increase in effluent COD compared with influent. The amounts of carbon release of ginkgo nut shell, walnut shell, and hazelnut shell were low (0.45, 0.41, and 0.43 mg COD g⁻¹, respectively). The released carbon could not be used easily by microorganisms. Meanwhile, the contents of degradable aromatic protein and protein-like in dissolved organic matter (DOM) were low. Even the fulvic acid–like with low biodegradability also appeared in the soaking solution of the hazelnut shell. The NO3⁻-N and total nitrogen aveage removal rates were low in these three fruit shells and showed the removals within the 54.10–57.25% range and 52.21%–54.24% range, respectively. The amount of carbon release of pistachio shell was lower than that of peanut shell. However, the released carbon of the former was more biodegradable than that of the latter. Moreover, the relative molecular mass of DOM was small, and the contents of aromatic protein and protein-like were much higher than those of the four other kinds of fruit shells. The NO3⁻-N removal rate (71.48% ± 0.98%) of pistachio shell was only slightly lower than that of peanut shell. In conclusion, pistachio shell was the best carbon source for biological denitrification in the advanced treatment.
Glycerol is a by-product generated in the biodiesel industry and can be converted by biological processes into products such as hydrogen, alcohols and acids. This study aimed at comparing two sources of inoculum in dark fermentation using raw glycerol and at identifying the microorganisms present by high-performance sequencing. Granular sludge obtained from a vegetable oil industry (VOI) and anaerobic sludge from a wine industry (WI) were evaluated in batch reactors with raw glycerol. The highest hydrogen yield was obtained for VOI (1.12 mol H2 / mol glycerolconsumed). The WI was more efficient in producing soluble metabolites, with 6.2 g / L of 1,3 – propanediol, ethanol (2.5 g / L) and acids (122 mg / g glycerolconsumed). Both assays consumed more than 60% of the glycerol. A significant variation in microbial diversity was observed between the beginning and the end of the fermentation. The microbial community showed a predominance of Firmicutes and the Clostridiaceae and Peptostreptococcaceae families in different abundances among the inocula, which could lead to an increase in hydrogen production or a higher concentration of metabolites. Clostridium bifermentans was the only species isolated, showing a yield of H2 (0.135 mol H2 / mol glycerolconsumed) and the main metabolite produced was ethanol (670 mg / L). These results showed that two different kinds of seed sludge can lead to different metabolic profiles from the same substrate, highlighting the importance of choosing the inoculum and, consequently, that the knowledge regarding microorganisms can direct the process towards products of industrial interest and contribute to the generation of sustainable energy.
In order to evaluate the effect of sugarcane bagasse (SCB) heterogeneity and hydrothermal (HT) pretreatment on biofuels production, industrial and laboratorial, rind and pith, hydrothermally pretreated (200 °C for 10 min at 16 bar and 10 g of SCB mixed with 100 mL of water) and untreated sugarcane bagasse (SCB) were used as substrate (10 g/L) in batch reactors at thermophilic condition (55 °C) and initial pH of 6.0. High cellulose content in the hydrothermally (HT) pretreated sugarcane bagasse (SCB) results in hydrogen production (33.7 mmol/L), while lactic acid was the main metabolite produced using laboratorial untreated SCB rind and pith (1.5 and 1.3 g/L, respectively), which caused a decrease in the pH and no hydrogen production. Methane was only observed when industrial untreated SCB pith and rind (5.5 and 3.1 mmol/L), respectively, was used. 16S rRNA sequencing was carried out and at the end of the operation the reactors were fed with untreated laboratorial rind, hydrothermally pretreated industrial rind and untreated industrial pith. Hydrogen-producing bacteria, such as Acetomicrobium (74.4 %) was favored using the HT pretreated fiber, while lactic acid bacteria such as Lactococcus (6.3 %) had a potentially negative effect on the hydrogen-producing bacteria like Thermoanaerobacterium (75.0 %) using laboratorial untreated SCB, since no hydrogen was obtained in this condition. Microorganisms involved in all the anaerobic digestion steps, such as Clostridium (34.4 %), Acetomicrobium (33.4 %), and Methanoculleus (14.8 %) lead to hydrogen, volatile fatty acids (VFA), ethanol, and methane production using industrial untreated SCB. Therefore, this study successfully dissected the detailed microbial community structure and the shift on metabolic pathways as a response of SCB heterogeneity and hydrothermal pretreatment.
Nitrogen removal from the rural domestic sewage with low pollutant concentration and low chemical oxygen demand (COD) to total nitrogen (TN) ratio (C/N) by biological process is quite difficult. Raw broken Arundo donax (A. donax) pieces were supplied as anoxic column filter media and carbon source in the anoxic/oxic biofilter (hereinafter referred to as A.-A/O-BF) for the nitrogen removal from rural domestic sewage, while gravel was served as the anoxic column filter media in the control A/O biofilter (hereinafter referred to as G-A/O-BF). Under the condition of 3.34 m/d (anoxic column) and 1.35 m/d (oxic column) filtration rate, 4:1 air/water ratio (oxic column) and 150 % nitrate recycling ratio, the average NH4⁺-N and TN removal efficiency of A.-A/O-BF were 99.0 % and 68.8 %, which were much higher than those (91.4 % and 39.8 %) of G-A/O-BF. The class Nitrospira affiliated with the phylum Nitrospirae were the most widely distributed nitrifying bacteria in the oxic column of A.-A/O-BF, which rarely existed in the oxic column of G-A/O-BF. Dechloromonas and Chlorobium genera were the main denitrificans in the anoxic column of A.-A/O-BF, while SM1A02 and Denitratisoma were dominant in the anoxic column of G-A/O-BF. The abundance of the main nitrification functional genes (amoA and Nitrospira 16S rDNA) in the oxic column of A.-A/O-BF was two orders of magnitude higher than that of G-A/O-BF, and the denitrification functional genes (narG, nosZ, nirS, and nirK) and anaerobic ammonium oxidation functional gene (Anammox) in the anoxic column of A.-A/O-BF were also remarkably higher than those of G-A/O-BF.
Solid waste (pulp and husk) and wastewater from post-harvest coffee processing contain high carbohydrate content, which can be used in biofuel production. However, the recalcitrance of lignocellulosic material in the pulp and husk, in addition to the polyphenols in wastewater limits its application. The objective of this research was to evaluate the coffee processing waste for H2 production. The experiments were carried out in batch reactors analyzing the co-digestion of: (1) wastewater with pulp and husk in natura or ground, (2) pulp and husk pretreated in the hydrothermal system and (3) liquid fraction from pretreatment of the pulp and husk in the hydrothermal system. The severity factor of the pulp and husk hydrothermal pretreatment was between 3.2 and 4.2, in addition to bioaugmentation of the autochthonous consortium (bacteria and fungi) from the waste. The highest H2 production potential of 8 mL was obtained by co-digestion of pretreated pulp and husk in severity 3.5 with coffee processing wastewater. Pulp and husk pretreatment with the hydrothermal system at 180 °C for 15 min favored in increased 20% cellulose, 14% hemicellulose, and 31% lignin. Hydrothermal pretreatment and waste co-digestion improved up to 7 times the H2 production when compared to in natura waste.
The objective of this study was to evaluate the fermentation conditions that led to the optimization of H2 production from coffee waste (wastewater, pulp and husk) and the taxonomic and functional characterization of autochthonous microorganisms. Assays in batch reactors with microbial consortium bioaugmentation (bacteria and fungi) evaluated the pH (4.82–8.18), pulp and husk concentration (6.95–17.05 g/L) and headspace factor (33.18–66.82%) by means of rotational central composite design and response surface. Operating conditions in the reactor optimized for 3.04 LH2/Ld were at pH 7.0, 7 g/L pulp and husk and 30% headspace. The main metabolites observed were butyric acid (3838 mg/L), isobutyric acid (506 mg/L), methanol (226 mg/L) and butanol (156 mg/L). Clostridium sp. (87.9%), Lactobacillus sp. (1.7%), Kazachstania sp. (18.6%) and Saccharomyces sp. (16.3%) were the main genera identified in the optimized reactor, which had functional gene diversity for H2 production, alcoholic fermentation, cellulose degradation, lignin, hemicellulose and phenol.
The heat-shock pretreatment (HST) is a useful method to select for H2-producing inocula when soluble substrates are employed. However, the HST has proven to have negative effects on the H2 production performance from lignocellulosic substrates. We hypothesize that the negative effect of HST on H2 production from lignocellulosic substrates is due to the loss of species involved in cellulose solubilization. In the present study, we tested this hypothesis by applying a heat-shock pretreatment (105 °C/24 h) on the microbial community for producing hydrogen from microcrystalline cellulose. Specifically, we compared a microbial community treated with 2-bromoethanesulfonate (BES-treated control) versus a heat-shock pretreated microbial community. For both experimental treatments, we determined the major fermentation products (hydrogen, acetic, butyric, propionic, and isovaleric acids), as well as the diversity of bacteria and fungi using Illumina MiSeq of amplicons in five sampling points. We found that HST immediately reduced alpha diversity of microbial communities, being fungi more affected than bacteria. We also found that the bacterial reduction in Comamonas, Ureibacillus, and Aneurinibacillus was related to a low hydrogen production in the heat-shock pretreated community. Strictly anaerobic fungi such as Orpinomyces, Cyllamyces, and Neocallimastix, which are recognized by their role in solubilization of fibrous materials, were unable to survive the HST. The reconstructed bacterial network predicted positive interactions between cellulase-producing and hydrogen-producing families. We conclude that the HST did not promote the high microbial diversity required for hydrogen production from cellulose.
The molecular mechanisms behind the bioconversion of sugarcane bagasse into biofuel by promising metabolic pathways were studied, suggesting that proteolytic, cellulolytic and methanogenic microorganisms such as Coprothermobacter, Clostridium, and Methanothermobacter, respectively, took an important syntrophic role in lignocellulosic-derived fuel production. The mixed acid fermentation was the main route to the acetic, formic, butyric, and propionic acid production by acid-forming bacteria. Some aspects of biotechnological application of such metabolic pathways were evaluated from a central composite design, in which the effect of incubation temperature (from 45.8 to 74.2 °C) and yeast extract concentration (from 0.58 to 3.42 g/L) on hydrogen production were assessed. The interaction between these factors significantly affected the hydrogen production, which reached the highest value (17.3 mmol/L) using 3.42 g/L of yeast extract at 60 °C, and favored a plastic and physiological diverse microbial community related to bioconversion of SCB.
Sugarcane bagasse (SCB) was used as lignocellulosic substrate, combining the co-production of H2 (stage I) and CH4 (stage II) by dark fermentation process in batch reactors. Hydrothermally and enzymatic (Aspergillus niger) pretreatead SCB were applied as substrate sources. Two fermentative inocula (In1 and In2) were used in stage I and a methanogenic inoculum in stage II (In3), being in total three experimental series in relates to stage I: A (In1), B (In1 plus In2) and C (In2). The final metabolites from stage I were transferred to a second reactor for CH4 production (stage II). The SCB pretreatment employed was favorable for biogas and organic acids production. The higher H2 and CH4 yields were in C (4.3 and 6.3 mmol/g SCB, respectively). For all conditions, the H2 production occurred primarily via acetic acid route. Predominance of cellulolytic enzymes producers (Enterococcus and Clostridium) may have favored the H2 and subsequent CH4 production; this last produced mainly from members of Methanoregulaceae and Methanosaetaceae families. Furthermore, homoacetogenic bacteria (Acetobacterium, Clostridium, Eubacterium, Holophaga) were also identified. The synergistic action of these microbial groups promoted the hydrolysis of SCB and hydrogen and methane production.
Hydrogen can be produced through different methods. Various biomass can be used as low-cost substrate for fermentative hydrogen production, which significantly reduces the hydrogen production cost. Furthermore, biohydrogen production from biomass wastes can achieve dual benefits of clean energy generation and waste management since agricultural and municipal wastes can be disposed at the same time. However, the application of hydrogen production from biomass meets the bottlenecks of low hydrogen production rate and substrate degradation rate. In this paper, various biomass as feedstock, including waste activated sludge produced form wastewater treatment plant, algae, agricultural residuals and municipal wastes used for biological hydrogen production, was reviewed. Since the hydrolysis to smaller molecules is the rate-limiting step for biomass degradation, a pretreatment step can enhance both the hydrogen production efficiency and biomass degradation rate. Pretreatment process can destroy the crystal structure of macromolecular substances and reduce their polymerization degree. Therefore the trapped components can be released through cell wall lysis and delignification of lignocellulosic biomass to make higher proportion of readily fermentable substances accessible for microorganisms. Various pretreatment methods used for treating biomass as feedstock for hydrogen production were analyzed and compared. Physical treatment, chemical treatment, biological treatment and a combination of different treatments are usually used for the pretreatment of biomass. Physical treatment methods include mill, grind, ultra-sonication, heat, freeze and thaw, microwave and ionizing radiation; chemical treatment methods comprise acid and alkaline treatment, oxidation by oxidizing agent and addition of methanogenic inhibitors; biological treatment methods mainly consist of enzymatic treatment and bacterial hydrolysis. Pretreatment is a critical process for fermentative hydrogen production from biomass. Considerable efforts are needed from both technical and managing aspects to achieve a full-scale application of fermentative hydrogen production from biomass.
Hydrogen production from hydrothermally pretreated (200 WC for 10 minutes at 16 bar) sugarcane
bagasse was analyzed using response surface methodology. The yeast extract concentration and the
temperature had a significant influence for hydrogen production (p-value 0.027 and 0.009,
respectively). Maximum hydrogen production (17.7 mmol/L) was observed with 3 g/L yeast extract at
60 WC (C10). In this conditions were produced acetic acid (50.44 mg/L), butyric acid (209.71 mg/L),
ethanol (38.4 mg/L), and methane (6.27 mmol/L). Lower hydrogen productions (3.5 mmol/L and
3.9 mmol/L) were observed under the conditions C7 (2 g/L of yeast extract, 35.8 WC) and C9 (1 g/L of
yeast extract, 40 WC), respectively. The low yeast extract concentration and low temperature caused a
negative effect on the hydrogen production. By means of denaturing gradient gel electrophoresis
20% of similarity was observed between the archaeal population of mesophilic (35 and 40 WC) and
thermophilic (50, 60 and 64 WC) reactors. Likewise, similarity of 22% was noted between the bacterial
population for the reactors with the lowest hydrogen production (3.5 mmol/L), at 35.8 WC and with the
highest hydrogen production (17.7 mmol/L) at 60 WC demonstrating that microbial population
modification was a function of incubation temperature variation.
This study investigated the recovery of H2 and CH4 from bagasse bioethanol fermentation residues (bagasse BEFR) using a two-stage bioprocess. In the hydrogen fermentation bioreactor (HFB), carbohydrate removal efficiency was maintained at 82–93% and the highest hydrogen yield was 8.24 mL/g COD at volumetric loading rate (VLR) of 80 kg COD/m3/day. The results indicated a positive correlation between hydrogen yield and butyrate-to-acetate ratio, which might be due to the mechanisms of lactate/acetate utilization for hydrogen production and acetogenesis occurring in the HFB. Remaining volatile fatty acids and alcohols in the HFB effluent were further utilized for methane production in methane fermentation bioreactor (MFB), in which the highest methane yield of 345.2 mL/g COD was attained at VLR of 2.5 kg COD/m3/day. Overall, the two-stage bioprocess achieved a maximum COD removal of 81% from bagasse BEFR, and converted 0.3% and 72.8% of COD in the forms of H2 and CH4, respectively.
There is a pressing need for renewable and optimal use of resources towards sustainable primary production and processing systems worldwide. Current technologies for food and feedstock production are held accountable for several environmental problems, such as for instance soil and water contamination due to the use of hazardous substances, generation of toxic products and even excess of biomass that is considered waste. To minimize or solve these questions in order to produce an adequate quantity of reliable and healthy food, fibers and other products and energy, new paradigms focusing on sustainable agriculture, bio-based industries or biorefineries have emerged over the last decades. Biorefineries integrate sustainable and environmentally friendly concepts of Green Chemistry with intelligent and integrated farming processes, optimizing the agricultural production. Thermochemical and biochemical processes are excellent alternatives for the production of new classes of renewable biofuels and feedstock, showing relatively small impact on greenhouse gas emissions and important pathways to obtain platform chemicals. This review discusses the current and incipient technological developments for using biomass to generate bio-based chemicals over the last decade, focusing on Green Chemistry concepts towards sustainable agriculture and processing models in Brazil.
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.
Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention
because its combustion releases only water, and it can be produced by microorganisms
using organic acids as substrates. The aim of this study was to enrich a microbial
consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge
Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other
carbon sources were tested, such as acetate (30 mmol l�1), butyrate (17 mmol l�1), citrate
(11 mmol l�1), lactate (23 mmol l�1) and malate (14.5 mmol l�1). The reactors were incubated
at 30 �C under constant illumination by 3 fluorescent lamps (81 mmol m�2 s�1). The
cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H2 l�1 culture for
acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield
was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H2 mmol�1 substrate for acetate, butyrate, citrate, lactate
and malate, respectively. The consumption of substrates was 43% for acetate, 37% for
butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the
clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC)
were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely
cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum
(29% of the total) were also found in the microbial consortium.
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.
The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism of glucose fermentation and hydrogen production performance of four Clostridial species, including C. acetobutylicum M121, C. butyricum ATCC19398, C. tyrobutyricum FYa102, and C. beijerinckii L9. Batch experiments showed that all the tested strains fermented glucose, reduced medium pH from 7.2 to a value between 4.6 and 5.0, and produced butyrate (0.37–0.67 mmol/mmol-glucose) and acetate (0.34–0.42 mmol/mmol-glucose) as primary soluble metabolites. Meanwhile, a significant amount of hydrogen gas was produced accompanied with glucose degradation and acid production. Among the strains examined, C. beijerinckii L9 had the highest hydrogen production yield of 2.81 mmol/mmol-glucose. A kinetic model was developed to evaluate the metabolism of glucose fermentation of those Clostridium species in the batch cultures. The model, in general, was able to accurately describe the profile of glucose degradation as well as production of biomass, butyrate, acetate, ethanol, and hydrogen observed in the batch tests. In the glucose re-feeding experiments, the C. tyrobutyricum FYa102 and C. beijerinckii L9 isolates fermented additional glucose during re-feeding tests, producing a substantial amount of hydrogen. In contrast, C. butyricum ATCC19398 was unable to produce more hydrogen despite additional supply of glucose, presumably due to the metabolic shift from acetate/butyrate to lactate/ethanol production.
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.
Author Summary
Leaf-cutter ants form massive subterranean colonies containing millions of workers that harvest hundreds of kilograms of leaves each year. They use these leaves to grow a mutualistic fungus that serves as the colony's primary food source. By farming fungus in specialized garden chambers, these dominant Neotropical herbivores facilitate rapid large-scale plant biomass conversion. Our understanding of this degradation process, and the responsible microbial community, is limited. In this study, we track the degradation of plant polymers in leaf-cutter ant fungus gardens and characterize the microbial community potentially mediating this process. We show that cellulose and hemicelluloses are degraded in the fungus gardens and that a previously unknown microbial community containing a diversity of bacteria is present. Metagenomic analysis of this community's genetic content revealed many genes predicted to encode enzymes capable of degrading plant cell walls. The ability of leaf-cutter ants to maintain an external microbial community with high plant biomass-degrading capacity likely represents a key step in the establishment of these ants as widespread, dominant insect herbivores in the Neotropics. This system is an important model for understanding how microbial communities degrade plant biomass in natural systems and has direct relevancy for bioenergy, given recent interest in cellulosic biofuels.
The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.
Background:
Economic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.
Methodology/principal findings:
In this study, we used quantitative proteomics (multidimensional LC-MS/MS and (15)N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to (15)N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.
Conclusions/significance:
Overall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.
The ability of fungi to degrade lignocellulosic materials is due to their highly efficient enzymatic system. Fungi have two types of extracellular enzymatic systems; the hydrolytic system, which produces hydrolases that are responsible for polysaccharide degradation and a unique oxidative and extracellular ligninolytic system, which degrades lignin and opens phenyl rings. Lignocellulosic residues from wood, grass, agricultural, forestry wastes and municipal solid wastes are particularly abundant in nature and have a potential for bioconversion. Accumulation of lignocellulosic materials in large quantities in places where agricultural residues present a disposal problem results not only in deterioration of the environment but also in loss of potentially valuable material that can be used in paper manufacture, biomass fuel production, composting, human and animal feed among others. Several novel markets for lignocellulosic residues have been identified recently. The use of fungi in low cost bioremediation projects might be attractive given their lignocellulose hydrolysis enzyme machinery.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
In anaerobic environments rich in decaying plant material, the decomposition of cellulose is brought about by complex communities of interacting microorganisms. Because the substrate, cellulose, is insoluble, bacterial and fungal degradation occurs exocellularly, either in association with the outer cell envelope layer or extracellularly. Products of cellulose hydrolysis are available as carbon and energy sources for other microbes that inhabit environments in which cellulose is biodegraded, and this availability forms the basis of many microbial interactions that occur in these environments. This review discusses interactions among members of cellulose-decomposing microbial communities in various environments. It considers cellulose decomposing communities in soils, sediments, and aquatic environments, as well as those that degrade cellulose in association with animals. These microbial communities contribute significantly to the cycling of carbon on a global scale.
Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.
Production of hydrogen by anaerobes, facultative anaerobes, aerobes, methylotrophs, and photosynthetic bacteria is possible. Anaerobic Clostridia are potential producers and immobilized C. butyricum produces 2 mol H2/mol glucose at 50% efficiency. Spontaneous production of H2 from formate and glucose by immobilized Escherichia coli showed 100% and 60% efficiencies, respectively. Enterobactericiae produces H2 at similar efficiency from different monosaccharides during growth. Among methylotrophs, methanogenes, rumen bacteria, and thermophilic archae, Ruminococcus albus, is promising (2.37 mol/mol glucose). Immobilized aerobic Bacillus licheniformis optimally produces 0.7 mol H2/mol glucose. Photosynthetic Rhodospirillum rubrum produces 4, 7, and 6 mol of H2 from acetate, succinate, and malate, respectively. Excellent productivity (6.2 mol H2/mol glucose) by co-cultures of Cellulomonas with a hydrogenase uptake (Hup) mutant of R. capsulata on cellulose was found. Cyanobacteria, viz., Anabaena, Synechococcus, and Oscillatoria sp., have been studied for photoproduction of H2. Immobilized A. cylindrica produces H2 (20 ml/g dry wt/h) continually for 1 year. Increased H2 productivity was found for Hup mutant of A. variabilis. Synechococcus sp. has a high potential for H2 production in fermentors and outdoor cultures. Simultaneous productions of oxychemicals and H2 by Klebseilla sp. and by enzymatic methods were also attempted. The fate of H2 biotechnology is presumed to be dictated by the stock of fossil fuel and state of pollution in future.
A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates
concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction.
Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from
the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.
Mobility--the transport of people and goods - is a socioeconomic reality that will surely increase in the coming years. It should be safe, economic and reasonably clean. Little energy needs to be expended to overcome potential energy changes, but a great deal is lost through friction (for cars about 10 kWh per 100 km) and low-efficiency energy conversion. Vehicles can be run either by connecting them to a continuous supply of energy or by storing energy on board. Hydrogen would be ideal as a synthetic fuel because it is lightweight, highly abundant and its oxidation product (water) is environmentally benign, but storage remains a problem. Here we present recent developments in the search for innovative materials with high hydrogen-storage capacity.
On the basis of 16S rRNA gene sequencing and DNA-DNA reassociation, industrial solvent-producing clostridia have been assigned to four species. In this study, the phenotypic characteristics of Clostridium acetobutylicum, Clostridium beijerinckii, 'Clostridium saccharoperbutylacetonicum', and an unnamed Clostridium sp. represented by the strains NCP 262T and NRRL B643 are compared. In addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, DNA fingerprinting and 16S rRNA gene sequencing. These included 14 C. beijerinckii strains, two strains currently designated as 'Clostridium kaneboi' and 'Clostridium butanologenum', and 24 production strains used in the commercial acetone-butanol fermentation. All of the C. beijerinckii strains were confirmed to have been classified correctly. The 'C. kaneboi' and 'C. butanologenum' strains require reclassification as C. acetobutylicum and C. beijerinckii, respectively. The commercial production strains were found to belong either to C. beijerinckii or to the unnamed Clostridium sp. For the comparative phenotypic studies of the four species, representative strains were selected from each of the DNA-fingerprint subgroups within each species. These strains were analysed for their ability to utilize different carbohydrates, hydrolyse gelatin or aesculin, and produce indole, and were tested for the presence of catalase and urease. On the basis of these results, several phenotypic traits were found to be useful for differentiating between the four species. The descriptions of C. acetobutylicum and C. beijerinckii have been emended. The names Clostridium saccharoperbutylacetonicum sp. nov. [type strain = N1-4 (HMT) = ATCC 27021T] and Clostridium saccharobutylicum sp. nov. (type strain = DSM 13864T = ATCC BAA-117T) are proposed for the two new species.
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.
Hydrogen production was obtained from low concentrations of xylose metabolized by heat treated inoculum obtained from the slaughterhouse wastewater treatment UASB reactor installed in Brazil. The molecular biological analysis Clostridium and Klebsiella species, recognized as H2 and volatile acid producers, in addition to Burkholderia species and uncultivated bacteria. The assays were carried out in batch reactors: (1) 630.0 mg xylose/L, (2) 1341.0 mg xylose/L, (3) 1848.0 mg xylose/L and (4) 3588.0 mg xylose/L. The following yields were obtained: 3% (0.2 mol H2/mol xylose), 8% (0.5 mol H2/mol xylose), 10% (0.6 mol H2/mol xylose) and 14% (0.8 mol H2/mol xylose), respectively. The end products obtained were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. The concentrations of xylose did not inhibit microbial growth and hydrogen production. This suggested that low concentrations of xylose should be added to wastewater to produce hydrogen.
The effects of pH and moisture content on high-solids sludge digestion were investigated using a mesophilic batch digester fed with sludge cake. The experiments were carried out by changing the initial moisture contents from 90 to 96% and the initial pH from 5.0 to 10.0. A simple model developed from the Gompertz equation was applied to estimate the methane production rate and the lag-phase time under various conditions, based on the cumulative methane production curves. The relative methanogenic activity decreased with the decrease of moisture content and dropped from 100 to 53% when the moisture content decreased from 96 to 90%. The rate of the methane production of the high-solids digestion at moisture contents of 90 to 96% functioned over a range of 6.6–7.8 with an optimum of pH 6.8, whereas the process may fail if the pH is lower than 6.1 or higher than 8.3. Moreover, a minimum lag-phase time for methane production was also found at around pH 6.8. In addition, a modified Haldane equation was suitable to represent the effect of pH on methanogenic activity at various moisture contents. The maximum specific methane production rate, Km, and the saturation rate constants for hydrogen ion, KH, and hydroxyl ion, KOH, were 14.5-7.5 ml CH . g−1 dry weight . d−1, 5.0 × 10−1–8.0 × 10−5 [M] and 2.0 × 10−9–8.0 × 10−9 [M], respectively.
Pre-heated elephant dung was used as inoculum to produce hydrogen from sugarcane bagasse (SCB) hydrolysate. SCB was hydrolyzed by H2SO4 or NaOH at various concentrations (0.25–5% volume) and reaction time of 60 min at 121 °C, 1.5 kg/cm2 in the autoclave. The optimal condition for the pretreatment was obtained when SCB was hydrolyzed by H2SO4 at 1% volume which yielded 11.28 g/L of total sugar (1.46 g glucose/L; 9.10 g xylose/L; 0.72 g arabinose/L). The maximum hydrogen yield of 0.84 mol H2/mol total sugar and the hydrogen production rate of 109.55 mL H2/L day were obtained at the initial pH 6.5 and initial total sugar concentration 10 g/L. Hydrogen-producing bacterium (Clostridium pasteurianum) and non hydrogen-producing bacterium (Flavobacterium sp.) were dominating species in the elephant dung and in hydrogen fermentation broth. Sporolactobacillus sp. was found to be responsible for a low hydrogen yield obtained.
Lignocellulosic biomass contains 70–80% carbohydrates and could serve as the ideal feedstock for fermentative hydrogen production. We conducted the pretreatment of corn stover using a steam-explosion process and studied its fermentability for hydrogen production. Using natural inoculant obtained from the heated sludge of a local wastewater treatment plant, we demonstrated that the indigenous microbes were capable of efficiently fermenting the aqueous hydrolyzate derived from the hemicellulose fraction of the steam-pretreated corn stover with and without acid during pretreatment. Biogas contained equal amounts of hydrogen and carbon dioxide. The carbon mass balance is approximately 84%, with acetic and butyric acids as the major carbon byproducts along with carbon dioxide. Hydrogen molar yields of 2.84 and 3.0 were obtained using the mixed sugars present in the hydrolyzate derived from neutral and acidic steam explosion, respectively. These findings verify that hemicellulose from corn stover could be a suitable feedstock for hydrogen production via dark fermentation.
The optimization of total solids in the feed (%TS) and alkalinity ratio (γ) for H2 production from organic solid wastes under thermophilic regime was carried out using response surface methodology based on a central composite design. The total solids levels were 20.9, 23.0, 28.0, 33.0 and 35.1% whereas the levels of alkalinity ratio (defined as g phosphate alkalinity/g dry substrate) were 0.15, 0.20, 0.30, 0.41 and 0.45. High levels of TS and γ affected in a negative way the H2 productivity and yield; both response variables significantly increased upon decreasing the TS content and alkalinity ratio. The highest H2 productivity and yield were 463.7NmL/kg-d and 54.8NmL/gVSrem, respectively, predicted at 20.9% TS and alkalinity ratio 0.25 (0.11gCaCO3/g dry substrate). The alkalinity requirements for hydrogenogenic processes were lower than those reported for methanogenic processes (0.11 vs. 0.30gCaCO3/gCOD). Adequate alkalinity ratio was necessary to maintain optimal biological activity for hydrogen production; however, excessive alkalinity negatively affected process performance probably due to an increase of osmotic pressure. Interestingly, reactor pH depended only on the alkalinity ratio, thus the buffer capacity was able to maintain a constant pH independently of TS levels. At γ=0.15–0.30 the pHs were in the range 5.56–5.95, which corresponded to the highest hydrogen productivities and yields. Finally, the highest metabolite accumulation corresponded with the highest removal efficiencies but not with high H2 productivities and yields. Therefore, it seems that organic matter removal was channeled toward solvent generation instead of hydrogen production at high TS and γ levels. This is the first study that shows the requirements of alkalinity in solid substrate fermentation conditions for H2 production processes and their interaction with the content of total solids in the feed.
Experimental conditions are presented for continuous fermentation of xylose and glucose to hydrogen (H2), using Clostridium sp. strain No. 2 growing in 300 mL of culture containing 0.3% substrate, either without pH control or with the pH controlled at 6.0. The H2 production rate increased in proportion to increasing dilution rate within the range of about 0.4 to 1.0 h−1. The maximal H2 production rates of 21.03 and 20.40 mmol∙h−1∙L−1 were obtained from xylose and glucose with dilution rates of 0.96 and 1.16 h−1, respectively, at pH 6.0. About 2.06 mol H2 with a dilution rate of 0.21 h−1 at pH 6.0 and 2.36 mol H2 with a dilution rate of 0.18 h−1 were formed per mole of xylose and glucose consumed, respectively, at uncontrolled pH.Key words: hydrogen production, continuous fermentation, xylose, Clostridium sp. strain no. 2.
For simultaneous saccharification and fermentation of cellulosic bioresources to biofuels, a stable rumen-mimicking bacterial consortium was established and maintained by sub-culturing and is referred to here as the “Functional Rumen Bacterial Consortium” (FRBC). The microbial community was analyzed by denaturing gradient gel electrophoresis, which contained dominant species such as Clostridium xylanolyticum, Clostridium papyrosolvens, Clostridium beijerinckii, Clostridium puniceum, Clostridium putrifaciens, and Ruminococcus sp. These predominant Clostridial strains showed potential in converting lignocellulosic material into bio-hydrogen and bio-fuels. The Clostridial strains with 16S rRNA sequences identical to the DGGE profile were isolated from FRBC. The isolate C. puniceum strain Ru6 exhibited xylanase and pectinase activity and higher hydrogen productivity and was thus chosen as the partner strain for ethanol production with C. xylanolyticum Ru15, which showed additional endoglucanase activity. A co-culture approach was used to reconstruct the FRBC with Ru6 and Ru15, and the productivities of hydrogen and ethanol were comparable to that of the FRBC. These results demonstrate the potential of a biologically inspired rumen-mimic microbial community to produce cellulosic bio-fuels.Highlights► The stable Functional Rumen Bacterial Consortium was established by sub-culturing. ► The microbial community was analyzed by denaturing gradient gel electrophoresis. ► The Clostridium strains Ru6 and Ru15 were sieved from FRBC. ► A co-culture approach was used to reconstruct the FRBC with Ru6 and Ru15. ► Establishment of rumen-mimic microbial community to produce cellulosic bio-fuels.
Sixteen batch experiments were performed to evaluate the stability, kinetics, and metabolic paths of heat-shocked digester (HSD) sludge that transforms microcrystalline cellulose into hydrogen. Highly reproducible kinetic and metabolic data confirmed that HSD sludge could stably convert microcrystalline cellulose to hydrogen and volatile fatty acids (VFA) and induce metabolic shift to produce alcohols. We concluded that clostridia predominated the hydrogen-producing bacteria in the HSD sludge. Throughout this study the hydrogen percentage in the headspace of the digesters was greater than 50% and no methanogenesis was observed. The results emphasize that hydrogen significantly inhibited the hydrogen-producing activity of sludge when initial microcrystalline cellulose concentrations exceeded 25.0 g/L. A further 25 batch experiments performed with full factorial design incorporating multivariate analysis suggested that the ability of the sludge to convert cellulose into hydrogen was influenced mainly by the ratio of initial cellulose concentration (So) to initial sludge density (Xo), but not by interaction between the variables. The hydrogen-producing activity depended highly on interaction of So × (So/Xo). Through response surface analysis it was found that a maximum hydrogen yield of 3.2 mmol/g cellulose occurred at So = 40 g/L and So/Xo = 8 g cellulose/g VSS. A high specific rate of 18 mmol/(g VSS-d) occurred at So = 28 g/L and So/Xo = 9 g cellulose/g VSS. These experimental results suggest that high hydrogen generation from cellulose was accompanied by low So/Xo. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 280–287, 2001.
Since hydrogen is a renewable energy source, biohydrogen has been researched in recent years. However, data on hydrogen fermentation
by a leachate from a waste landfill as inoculum are scarce. We investigated hydrogen production using a leachate from an industrial
waste landfill in Kanagawa Prefecture. The results showed no methane gas production, and the leachate was a suitable inoculum
for hydrogen fermentation. The maximum H2 yield was 2.67 mol of H2 per mol of carbohydrate added, obtained at 30°C and an initial pH of 7. The acetate and butyrate production was significant
when the H2 yield was higher. Oxidation–reduction potential analysis of the culture suggested that hydrogen-producing bacteria in the
leachate were facultative anaerobic. Scanning electron microscope observations revealed that the hydrogen-producing bacteria
comprised bacilli about 2μm in length.
KeywordsLeachate–Industrial waste landfill–Biohydrogen–Facultative anaerobic–ORP
The effect of substrate concentration on hydrogen production was investigated using a continuous-flow stirred-tank reactor (CSTR). Sucrose was used as a model substrate. The CSTR was started at a sucrose concentration of 30 g COD/L and exhibited stable H2 production for 271 days at inlet sucrose concentrations of 10–60 g COD/L. Hydrogen production depended on the substrate concentration such that the highest values of 1.09 mol H2/mol hexoseadded, 1.22 mol H2/mol hexoseconsumed, 7.65 L H2/L/d, and 3.80 L H2/g VSS/d were recorded at a sucrose concentration of 30 g COD/L. All bacterial species detected by polymerase chain reaction-denaturing gradient gel electrophoresis analysis were H2-producing Clostridium spp. At inlet sucrose concentrations below 20 g COD/L, the H2 yield per hexoseconsumed decreased along with a significant decrease in the n-butyrate/acetate ratio. At the same range of sucrose concentrations, Clostridium scatologenes (an H2-consuming acetogen) was found in the sludge. At inlet sucrose concentrations over 35 g COD/L, substrate overload occurred and caused a decrease in the carbohydrate degradation efficiency and H2 yield per hexoseadded.
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.
In this work, an integrated enzymatic hydrolysis and dark–photo fermentation were employed to enhance the performance of H2 production from starch feedstock. The starch feedstock was first hydrolyzed in sequencing batch reactor containing indigenous starch hydrolytic bacterium Caldimonas taiwanensis On1, producing reducing sugar at a yield and rate of 0.5 g reducing sugar/g starch and 1.17 g reducing sugar/h/L, respectively, under the optimal condition of pH 7.0, 55 °C and 1.0 vvm (air volume per reactor volume per minute) aeration rate. The hydrolyzed starch was continuously introduced to dark fermentation bioreactor, where the hydrolysate was converted to H2 at a rate of 0.22 L/h/L by Clostridium butyricum CGS2 at pH 5.8–6.0, 37 °C and 12 h HRT. The resulting effluent from dark fermentation became the influent of continuous photo H2 production process inoculated with Rhodopseudomonas palustris WP3-5 under the condition of 35 °C, 100 W/m2 irradiation, pH 7.0 and 48 h HRT. Combining enzymatic hydrolysis, dark fermentation and photo fermentation led to a marked improvement of overall H2 yield (up to 16.1 mmol H2/g COD or 3.09 mol H2/mol glucose) and COD removal efficiency (ca. 54.3%), suggesting the potential of using the proposed integrated process for efficient and high-yield bioH2 production from starch feedstock.
A series of batch tests were conducted to investigate the effects of pH and intermediate products on biological hydrogen production. The tests were run in serum bottles to determine the optimal operating conditions to maximize hydrogen production using sucrose and starch as organic substrates. Apart from hydrogen, variations in pH, volatile fatty acids, and solvent concentrations were also monitored. Initial pH was found to have a profound effect on both hydrogen production potential and hydrogen production rate. A mixed microbial culture was involved in the fermentation process with H2, propionate, acetate, butyrate, and CO2 as major products. The lowest initial pH of 4.5 gave the highest specific hydrogen production potentials of H2/g chemical oxygen demand (COD) and H2/g COD for sucrose and starch respectively, but with the lowest specific hydrogen production rate. Although hydrogen production started earlier with the high production rate at a higher initial pH, the duration of the production was shorter. The rapid pH depletion could have caused a metabolic alteration of the microorganisms involved in hydrogen production, thereby resulting in the shift of intermediates production pathway [variation of the acetate/butyrate (HAc/HBu) ratio] and a consequent decrease in hydrogen production. The specific hydrogen production rate was highest for the pH range of 5.5–5.7. For the optimum pH range, the HAc/HBu ratio was in the range of 3–4 for both sucrose and starch. The findings of this study can be applied in the design of a high rate hydrogen bioreactor.
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.
Bacterial community composition during steady-state, fermentative H2 production was compared across a range of organic loading rates (OLRs) of 0.5–19 g COD l−1 h−1 in a 2-l continuous flow reactor at 30 °C. The varied OLRs were achieved with glucose concentrations of 2.5–10 g l−1 and hydraulic retention times of 1–10 h. The synthetic wastewater feed was amended with l-cysteine and maintained at a pH of 5.5. For each run at a given glucose concentration, the reactor was inoculated with an aliquot of well-mixed agricultural topsoil that had been heat-treated to reduce numbers of vegetative cells. At OLRs less than 2 g COD l−1 h−1, DNA sequences from ribosomal RNA intergenic spacer analysis profiles revealed more diverse and variable populations (Selenomonas, Enterobacter, and Clostridium spp.) than were observed above 2 g COD l−1 h−1 (Clostridium spp. only). An isolate, LYH1, was cultured from a reactor sample (10 g glucose l−1 at a 10-h HRT) on medium containing l-cysteine. In confirming H2 production by LYH1 in liquid batch culture, lag periods for H2 production in the presence and absence of l-cysteine were 5 and 50 h, respectively. The 16S rRNA gene sequence of LYH1 indicated that the isolate was a Clostridium sp. affiliated with RNA subcluster Ic, with >99% similarity to Clostridium sp. FRB1. In fluorescent in situ hybridization tests, an oligonucleotide probe complementary to the 16S rRNA of LYH1 hybridized with 90% of cells observed at an OLR of 2 g COD h−1, compared to 26% of cells at an OLR of 0.5 g COD l−1 h−1. An OLR of 2 g COD l−1 h−1 appeared to be a critical threshold above which clostridia were better able to outcompete Enterobacteriaceae and other organisms in the mixed soil inoculum. Our results are discussed in light of other biohydrogen studies employing pure cultures and mixed inocula.
A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H2/L-medium and the volumetric hydrogen production rate at 172 mL H2/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H2/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H2/L-medium and 1034 mL H2/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.
A gram negative hydrogen producing facultative anaerobe was isolated and characterized as Enterobacter cloacae IIT-BT 08. Hydrogen yields by using this microorganism varied from substrate to substrate (2.2 mol/mol glucose, 6 mol/mol of sucrose and 5.4 mol/mol cellobiose considering 1% w/v substrate in MY medium). The maximum rate of hydrogen production achieved was at 36°C and initial pH 6.0. The maximum rate was 29.63 mmol/(g dry cell per h). The pH profiles of the fermentation broth under aerobic and anaerobic conditions were monitored and found to differ from each other particularly beyond the pH of 4.8. About 28% of substrate energy were recovered in the form of hydrogen using sucrose as a substrate.
Palm oil mill effluent (POME) sludge, sludge compost from Malaysia and CREST compost from Philippines were collected for the study. The capability of these microflora to produce hydrogen was examined with artificial wastewater containing 1% glucose, 0.2% yeast extract and 0.018% magnesium chloride hexahydrate under anaerobic fermentation in a batch culture. The microflora in POME sludge, sludge compost and CREST compost were found to produce significant amounts of hydrogen. The maximum production yield of hydrogen per decomposed glucose was -glucose at a conversion rate of at 50°C obtained by sludge compost. All fermentations were carried out without pH control. It was also found that the addition of nitrogen source in the medium caused a change in hydrogen produced. There was no methane gas in the evolved gas.
An aciduric facultative anaerobe with a hydrogen-producing ability was isolated and identified as Enterobacter aerogenes strain HO-39. The bacterium was able to grow at acidic pH of 3.3 aerobically and at 4.0 anaerobically. Although the optimum pH for hydrogen production was 6.0 to 7.0, hydrogen could be produced at acidic pH of 4.0. The optimum temperature for hydrogen production and cell growth was 38°C. Among various carbohydrates, glucose was suited for hydrogen production and the yield was 1.0 mol-H2/mol-glucose. Polypepton was a favorable nitrogen source and hydrogen production was suppressed in a medium where glucose and Polypepton were autoclaved simultaneously. In a continuous culture, stable hydrogen production without the need for pH control in a reactor was attained under the condition where pH of the culture broth was more than 4.5.
Simple sugars, oligosaccharides, polysaccharides, and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange-yellow color when treated with phenol and concentrated sulfuric acid. The reaction is sensitive and the color is stable. By use of this phenol-sulfuric acid reaction, a method has been developed to determine submicro amounts of sugars and related substances. In conjunction with paper partition chromatography the method is useful for the determination of the composition of polysaccharides and their methyl derivatives.
During the process of papermaking by pulp and paper plants, a thick and viscous deposits, termed slime, is quickly formed around the paper machines, which can affect the papermaking process. In this study, we explored the composition of the bacterial biota in slime that developed on shower pipes from 2 machines at a Canadian paper mill. Firstly, the composition was assessed for 12 months by DNA profiling with polymerase chain reaction coupled with denaturing gradient gel electrophoresis. Except for short periods (2-3 months), clustered analyses showed that the bacterial composition of the slime varied substantially over the year, with less than 50% similarity between the denaturing gradient gel electrophoresis profiles. Secondly, the screening of 16S rRNA gene libraries derived from 2 slime samples showed that the most abundant bacteria were related to 6 lineages, including Chloroflexi, candidate division OP10, Clostridiales, Bacillales, Burkholderiales, and the genus Deinococcus. Finally, the proportion of 8 bacterial lineages, such as Deinococcus sp., Meiothermus sp., and Chloroflexi, was determined by the Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization in 2 slime samples. The results showed a high proportion of Chloroflexi, Tepidimonas spp., and Schlegelella spp. in the slime samples.
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.
This research for the first time investigated hydrogen production from the fresh leachate originated from municipal solid wastes. We found that fermentation of the leachate generated H(2) and was very much enhanced in the presence of extra phosphate in the batch reactor. The continuous expanded granular sludge bed (EGSB) reactor started to generate H(2) at day 20 and continued to 176 days with 120 mg/l of extra phosphate present. The highest chemical oxygen demand (COD) removal efficiency (66.9%) was achieved at liquid up-flow velocity of 3.7 m/h and hydraulic retention time of 12h. Under proposed optimal operation conditions, the mean H(2) production rate reached up to 2155 ml/(l day). We also found that over 80% liquid metabolites were acetic acid and ethanol, suggesting the ethanol-type fermentation was dominant in the bioreactor. These findings indicate that the fresh leachate can be used as the source for continuous hydrogen production.
Cultivation-independent molecular approaches were used to investigate the phylogenetic composition of Archaea and the relative abundance of phylogenetically defined groups of methanogens in the leachate of a closed municipal solid waste landfill. Cloning and phylogenetic analysis of archaeal 16S rRNA gene sequences (16S rDNA) revealed that the landfill leachate harbored a diverse Archaea community, with sequence types distributed within the two archaeal kingdoms of the Euryarchaeota and the Crenarchaeota. Of the 80 clones examined, 51 were phylogenetically associated with well-defined methanogen lineages covering two major methanogenic phenotypes; 20 were related to Thermoplasma and were grouped with some novel archaeal rRNA gene sequences recently recovered from various anaerobic habitats; finally, five belonged to Crenarchaeota and were not closely related to any hitherto cultivated species. Most of the methanogen-like clones were affiliated with the hydrogenotrophic Methanomicrobiales and the methylotrophic and acetoclastic Methanosarcinales. Quantitative oligonucleotide hybridization experiments showed that methanogens in the leachate accounted for only a very small fraction of the total community (approximately 2%) and that Methanomicrobiales and Methanosarcinales constituted the majority of the total methanogenic population.
We analyzed the phylogenetic composition of bacterial community in the effluent leachate of a full-scale recirculating landfill using a culture-independent molecular approach. 16S rRNA genes were amplified directly from leachate DNA with universally conserved and Bacteria-specific rDNA primers and cloned. The clone library was screened by restriction fragment length polymorphism, and representative rDNA sequences were determined. Many bacterial sequences displaying relatively low levels of similarity to any other hitherto reported rDNA sequences were retrieved. A total of 103 bacterial sequence types were found in 195 analyzed clones. Roughly 90% of the sequence types were affiliated with low-G + C gram-positive bacteria, the Chlamydiae/Verrucomicrobia group and with the Cytophaga-Flexibacter-Bacteroides group, where the clone distribution was 53%, 21% and 19%, respectively. The other 10 sequence types represented 7% of the total clones, and they were either affiliated with well-recognized bacterial divisions Planctomycetes, Spirochaetes, Proteobacteria and Actinobacteria, or grouped within two recently proposed candidate divisions OP9 and OP11. The most frequent sequence type represented less than 10% of the total bacterial 16S rRNA gene sequences, and the 15 more frequent sequence types accounted for at least 47% of these sequences. Some rRNA gene sequences clustered with genera or taxa that were classically identified within anaerobic treatment systems. These results indicate that, despite recent expansion, our knowledge on the microbial diversity in anaerobic treatment systems is still limited.
A "temperature-shift" strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 degrees C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 degrees C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H(2) via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H(2)/g reducing sugar.
16S rRNA gene sequencing was performed on the species Lactobacillus minutus, Lactobacillus rimae and Streptococcus parvulus in order to clarify their taxonomic position. Based on comparative sequence analyses these organisms represent a hitherto unknown line of descent within the lactic acid group of bacteria for which a new genus, Atopobium gen. nov., is proposed.
Taxonomic studies were performed on an anaerobic Gram-positive, spore-forming, psychrophilic bacterium originally isolated from spoiled vacuum-packed refrigerated beef. Based on the present finding it is proposed that this unknown psychrophilic bacterium be classified as a new species of the genus Clostridium, as Clostridium estertheticum sp. nov. The type strain is NCIMB 12511.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
Accurate measurements of the hydrogen gas produced by Escherichia coli and Hafnia alvei pure cultures during glucose metabolism were performed under different growth conditions: stagnant, with magnetic stirring or with vibrational shaking. These measurements were carried out using an electrochemical hydrogen sensor based on a platinum-coated solid polymer electrolyte membrane (Pt-SPE). The results obtained were dependent on the hydrodynamic conditions of the growth, with greater hydrogen production being associated with the stagnant conditions. These measurements will eventually enable us to elucidate whether the pathway used for glucose metabolism is either strictly or mainly anaerobic and to modify experimental conditions so as to influence the reaction.