16S rRNA gene based analysis of the microbial diversity and hydrogen production in three mixed anaerobic cultures

To explore of role of microbial diversity and its functionality in commercial bioreactors, three anaerobic microbial communities from Ontario, Canada were characterized using 16S rRNA gene-based, clone library sequencing and terminal restriction fragment length polymorphism (T-RFLP) and compared with the hydrogen (H₂) and methane yields. The T-RFLP method showed more operational taxonomic units than the clone library sequence analysis; however, the two methods showed similar dominant species and relative diversity while Spearman's Rank correlation coefficient (r) values ranged from 0.82 to 0.91. The Chao 1 and Shannon-Wiener indices revealed that the cultures samples have highly diverse microbial communities. Comparatively, cultures from a municipal wastewater treatment plant (CA) showed more diversity than those from facilities treating effluents from a baby food processor and a brewery. Even though culture CA has the highest microbial diversity, low H₂ and methane production yield was attributed to the presence of sulphate reducers, propionate producers and a low percentage of methanogens. This study confirms that the selection of the source of mixed anaerobic cultures plays an important role in H₂ and methane production.     

Assessing the impact of palmitic,myristic and lauric acids on hydrogen production from glucose fermentation by mixed anaerobic granular cultures

The effects of lauric (LUA), myristic (MA), palmitic (PA), and a mixture of myristic:palmitic (MA:PA) acids on hydrogen (H₂) production from glucose degradation using anaerobic mixed cultures were assessed at 37 °C with an initial pH set at 5.0 and 7.131 mM of each acid. The maximum H₂ yield (2.53 ± 0.18 mol mol⁻¹ glucose) was observed in cultures treated with PA. A principal component analysis (PCA) of the by-products and the microbial population data sets detected similarities between the controls and PA treated cultures; however, differences were observed between the controls and PA treated cultures in comparison to the MA and LUA treated cultures. The flux balance analysis (FBA) showed that PA decreased the quantity of H₂ consumed via homoacetogenesis compared to the other LCFAs. The control culture was dominated by Thermoanaerovibrio acidaminovorans (60%), Geobacillus sp. and Eubacterium sp. (28%), while Clostridium sp. was less than 1%. Treatment with PA, MA, MA:PA, or LUA increased the H₂ producers (Clostridium sp. and Bacillus sp.) population by approximately 48, 67, 86, and 86%, respectively.     

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

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

Optimizing hydrogen production from a switchgrass steam exploded liquor using a mixed anaerobic culture in an upflow anaerobic sludge blanket reactor

In this study, the operation of an upflow anaerobic sludge blanket reactor (UASBR) producing hydrogen (H₂) from a steam-exploded switchgrass (SWG) liquor was statistically optimized. The factors consider included pH, hydraulic retention time (HRT) and linoleic acid (LA) concentration. Under optimal operational conditions (pH 5.0, 10 h HRT and 1.75 g L⁻¹ LA), which were close to the predicted conditions using the D-optimality index, the maximum H₂ and methane yield observed were 99.86 ± 5.6 mL g⁻¹ TVS and 0.5 ± 0.1 mL g⁻¹ TVS, respectively. Under maximum H₂-producing conditions, high levels of acetate plus butyrate were observed with low levels of ethanol and lactate. A principal component analysis revealed that clustering of the samples was based on the operating conditions and fermentation metabolites. The microbial profiles revealed that by lowering the HRT from 16 to 8 h or decreasing the pH from 7.0 to 5.0 in the controls caused a 50% reduction in the relative abundance of the terminal restriction fragments belonging to the methanogenic population (Methanobacteria, Methanomicrobia, Methanococci). With LA treatment, H₂ producers (Ruminococcaceae and Clostridiaceae) were dominant and methanogens were inhibited and/or washed-out from the UASBR.     

Feasibility of successive hydrogen and methane production in a single-reactor configuration of batch anaerobic digestion through bioaugmentation and stimulation of hydrogenase activity and direct interspecies electron transfer

Two-stage anaerobic digestion is a promising way to increase the efficiency of decomposition of organic waste. However, spatial separation of stages makes this technology less economically attractive. In this work, we studied a model of two-stage anaerobic digestion with stages separated in time in one reactor. To implement this model, we used a combination of (1) bioaugmentation with a hydrogen-producing Thermoanaerobacterium thermosaccharolyticum SP-H2, resistant to low pH, (2) setting a low initial pH (5.5) to reduce the activity of hydrogenotrophic methanogens, (3) supplementing additives in the form of soluble iron (II) and granular activated carbon (GAC) to stimulate hydrogenase activity and direct interspecies electron transfer (DIET), respectively. For comparison, two methanogenic inoculums with different dominant microbial groups and initial iron concentrations were used. With the simultaneous addition of GAC and iron (II), sequential production of hydrogen and methane was observed for both methanogenic inoculums. Without the addition of GAC, methane formation was practically not observed, which, apparently, indicated the impossibility of syntrophic degradation of acidogenesis products due to the high partial pressure of hydrogen and the absence of conductive material for activation of DIET. Significant differences in hydrogenase activity and the kinetics of hydrogen and methane formation according to the modified Gompertz equation depending on the iron content in the system may indicate the importance of this additive. Further studies should be aimed at determining the optimal initial pH, concentrations of stimulating additives, bioaugmentation and methanogenic cultures, as well as confirming the stability of the proposed single-reactor two-stage anaerobic digestion system in semi-continuous mode.     

High yield simultaneous hydrogen and ethanol production under extreme-thermophilic (70 °C) mixed culture environment

The effect of pH and medium composition on extreme-thermophilic (70 °C) dark fermentative simultaneous hydrogen and ethanol production (process performance and microbial ecology) was investigated. Hydrogen and ethanol yields were optimized with respect to glucose, peptone, FeSO₄, NaHCO₃, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations as well as initial pH as independent variables. A combination of low levels of both glucose (≤2 g/L) and vitamin solutions (≤1 mL/L) and high levels of initial pH (≥7), mineral salts solution (≥5 mL/L) and FeSO₄ (≥100 mg/L) stimulated the hydrogen production, while high level of glucose (≥5 g/L) and low levels of both initial pH (≤5.5) and mineral salts solution (≤1 mL/L) enhanced the ethanol production. High yield of simultaneous hydrogen and ethanol production (1.58 mol H₂/mol glucose combined with an ethanol yield of 0.90 mol ethanol/mol glucose) was achieved under extreme-thermophilic mixed culture environment. Results obtained showed that the shift of the metabolic pathways favouring either hydrogen or ethanol production was affected by the change in cultivation conditions (pH and medium composition). The mixed culture in this study demonstrated flexible ability for simultaneous hydrogen and ethanol production, depending on pH and nutrients formulation. The microorganisms involved could be regarded as simultaneous hydrogen/ethanol producers, as hydrogen and ethanol fermentation under all conditions was carried out by a group of extreme-thermophilic bacterial species related to Thermoanaerobacter, Thermoanaerobacterium and Caldanaerobacter.     

Firmicutes with iron dependent hydrogenase drive hydrogen production in anaerobic bioreactor using distillery wastewater

Distillery wastewater rich in organics is an inexpensive renewable resource for making first generation biofuel. Distillery wastewaters are mostly treated via the biomethanation route; however, in this study the conditions in sequential batch reactor (SBR) are being set to develop and analyze the microbial community that opted for hydrogen production. An optimum performance condition for a bioreactor was achieved after 40 days of operation, which gave substrate degradation rate of 0.72 kg/m³-day with volumetric hydrogen production of 0.32 mol H₂/m³-day. Study proposes that the dominant Delftia sp., a hydrogen oxidizing bacterium has been replaced during hydrogen production mode with dominant Anaerofilum sp., an anaerobic Firmicute and the iron dependent hydrogenases dominated as functional gene for hydrogen production. Future studies are required where process-engineering interventions could be applied to improve the hydrogen driving biochemical process.     

Effect of inhibitors on hydrogen consumption and microbial population dynamics in mixed anaerobic cultures

The impact of different chemical microbial stressors (2-bromoethanesulfonate (BES), furfural, fish oil, lauric acid (LUA) and linoleic acid (LA)) on the inhibition of mesophilic hydrogen (H₂) consumption was examined in this study. Hydrogen consumption half-life values were used to compare the extent of inhibition by the different microbial stressing agents. A statistical analysis of the percent H₂ consumed using Tukey's analysis revealed the following trend: Control > fish oil = linoleic acid (LA (C18:2)) = furfural > BES > lauric acid (LUA (C12:0). The terminal restriction fragment length polymorphism (T-RLFP) results indicated that aceticlastic methanogens (Methanosaeta sp., Methanosarcina sp.) and hydrogenotrophic methanogens (Methanococcus sp.) were inhibited by the different chemical stressing agents. Cultures fed LUA and LA had a high abundance of Clostridium sp., Clostridium propionicum and Propionibacterium acnes. In comparison, BES and furfural fed cultures contained large fractions of Clostridium sp., Eubacteria sp. and Bacteroides sp. while in the fish oil fed cultures, the dominant organism detected was Eubacteria sp. This study indicated that H₂ consumption was affected by the chemical stressing agent concentration.     

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 ± 0.07 mol H₂/mol glucose (mean ± S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 ± 0.03 mol H₂/mol glucose, 0.17 ± 0.01 mol H₂/mol glucose, 0.11 ± 0.01 mol H₂/mol glucose and 0.20 ± 0.04 mol H₂/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp.. However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community.     

Enhanced bio‐hydrogen production from sweet sorghum stalk with alkalization pretreatment by mixed anaerobic cultures

Bio‐hydrogen production from sweet sorghum stalk using mixed anaerobic sludge was reported in this paper. Batch tests were carried out to analyze influences of several environmental factors on yield of H₂ from sweet sorghum stalk under constant mesophillic temperature (36±1°C). The experimental results showed that, for the raw stalk, the cumulative hydrogen yield was 52.1 ml g⁻¹·TVS with utilization percentages of sugars, hemi‐cellulose and cellulose in the stalk being 89.12, 15.23 and 13.89% respectively; whereas for the stalk pretreated by 0.4% NaOH solution at room temperature for 24 h, the cumulative hydrogen yield was 127.26 ml g⁻¹·TVS with utilization percentages of sugars, hemi‐cellulose and cellulose being 99.17, 53.64, 41.56%, respectively. The hydrogen content in the biogas was about 53% while the methane content was less than 4% throughout the study. Besides hydrogen and methane, the main metabolic products detected were ethanol, propionate and butyrate. The experimental results suggested that the alkalization pretreatment of the substrate plays a crucial role in the conversion of the sweet sorghum stalk wastes into bio‐hydrogen by the mixed anaerobic sludge. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of different pretreatment methods on fermentation types and dominant bacteria for hydrogen production

In order to enrich hydrogen producing bacteria and to establish high-efficient communities of the mixed microbial cultures, inoculum needs to be pretreated before the cultivation. Four pretreatment methods including heat-shock pretreatment, acid pretreatment, alkaline pretreatment and repeated-aeration pretreatment were performed on the seed sludge which was collected from a secondary settling tank of a municipal wastewater treatment plant. In contrast to the control test without any pretreatment, the heat-shock pretreatment, acid pretreatment and repeated-aeration pretreatment completely suppressed the methanogenic activity of the seed sludge, but the alkaline pretreatment did not. Employing different pretreatment methods resulted in the change in fermentation types as butyric-acid type fermentation was achieved by the heat-shock and alkaline pretreatments, mixed-acid type fermentation was achieved by acid pretreatment and the control, and ethanol-type fermentation was observed by repeated-aeration pretreatment. Denaturing gradient gel electrophoresis (DGGE) profiles revealed that pretreatment method substantially affected the species composition of microbial communities. The highest hydrogen yield of 1.96 mol/mol-glucose was observed with the repeated-aeration pretreatment method, while the lowest was obtained as the seed sludge was acidified. It is concluded that the pretreatment methods led to the difference in the initial microbial communities which might be directly responsible for different fermentation types and hydrogen yields.     

Advances towards the understanding of microbial communities in dark fermentation of enzymatic hydrolysates: Diversity,structure and hydrogen production performance

Microbial communities involved in hydrogen (H₂) production from enzymatic hydrolysates of agave bagasse were analyzed through 16S rRNA sequencing. Two types of reactor configurations and four different enzymatic hydrolysates were evaluated. Trickling bed reactors led to highly-diverse microbial communities, but low volumetric H₂ production rates (VHPR, maximum: 5.8 L H₂/L-d). On the contrary, well-controlled environments of continuous stirred-tank reactors favored the establishment of low diverse microbial communities composed by Clostridium-Sporolactobacillus leading to high-performance H₂-production (VHPR maximum: 13 L H₂/L-d). Cellulase-Viscozyme and Celluclast-Viscozyme hydrolysates led to the co-dominance of Clostridium and Sporolactobacillus, possibly due to the presence of xylose and hemicellulose-derived carbohydrates. Cellulase hydrolysates were linked to communities dominated by Clostridium, while maintaining low abundance of Sporolactobacillus. Stonezyme hydrolysates favored microbial communities co-dominated by Clostridium-Lachnoclostridium-Leuconostoc. Moreover, contrary to the prevailing theory, it was demonstrated that H₂ production performance was inversely related to microbial diversity.     

Biohydrogen fermentation from Pistia stratiotes (aquatic weed) using mixed and pure bacterial cultures

In this study, mixed and pure bacterial cultures are inoculated in a dark-fermentation process for hydrogen production from Pistia stratiotes hydrolysate. The maximum hydrogen yield (HY) of 2.3 mol_H₂/mol_glucose (energy yield 0.3 kJ/g_DW) is obtained at pH 5.5, fermentation time 6.7 h, substrate-to-biomass ratio (S/X) 0.5 g_COD/g_VSS, and 25 °C using a mixed culture system. The plots of HY versus pH and time result in quadratic linear concave-up curves, whereas the effects of S/X and temperature on HY cause quadratic concave-up and flat curves, respectively. Fluorescent in situ hybridization (FISH) indicates that the mixed culture contains multiple H₂-producers including Enterobacteriaceae, Gammaproteobacteria, Betaproteobacteria, and Clostridium histolyticum. Three isolates belonging to Bacillus cereus, Bacillus anthracis, and Enterobacter cloacae are used as pure cultures, achieving HYs of 2.21, 1.10, and 1.97 mol_H₂/mol_glucose, respectively. Accordingly, the mixed culture inoculum subjected to thermal treatment at 100 °C for 30 min can deliver higher HYs than pure cultures.     

Flux balance analysis of mixed anaerobic microbial communities: Effects of linoleic acid (LA) and pH on biohydrogen production

The internal fluxes of mixed anaerobic cultures fed 2000 mg l⁻¹ linoleic acid (LA) plus glucose at 6 initial pH conditions and maintained at 37 °C were estimated using a flux balanced analysis (FBA). In cultures fed LA at pH 7, less than 8% of the flux was diverted to CH₄. At an initial pH ≥ 5.5, the quantity of glucose removed was greater than 95%; however, at pH 4.5 and 5.0 the quantity consumed were 38% and 75%, respectively. The FBA output showed that the acetogenic H₂-consumers were responsible for more than 20% of the H₂ consumed. Adding LA and decreasing the pH was ineffective in reducing the activity of acetogenic H₂-consumers. As the initial pH decreased, the acetogenic H₂-consuming flux decreased in the presence of 2000 mg l⁻¹ LA. A maximum H₂ yield of 1.55 mol mol⁻¹ glucose consumed (peak hydrogenase flux (R12)) was attained when the acetogenic H₂-consuming flux reached 0.42 mol at a pH of 5.5.     

Improving methane content and yield from rice straw by adding extra hydrogen into a two-stage anaerobic digestion system

This study was aimed to increase both the methane content and yield from rice straw by adding extra H₂ into a two-stage anaerobic digestion system. The results showed that the efficiency of H₂ utilization was 36.43%. Of the total amount of H₂ used, 47.3% was converted to CH₄, thereby increasing the CH₄ content and yield by 45% and 101%, respectively. This conversion was performed at a H₂/CO₂ ratio of 4/1 and a recycling frequency of two times a day in an up-flow reactor (UR). Due to the different ways of CH₄ production in continuously stirred tank reactor (CSTR) and UR, ethanol and acetate were produced as intermediaries, accounting for 65% and 87% of total intermediaries, respectively. Moreover, the pH values in CSTR and UR were 6.7 and 8.1, respectively. The results of the microbial community structures showed that the archaea genera of Methanosaeta and Methanobacterium were the most dominant in CSTR, whereas, Methanosaeta was the most dominant in UR. In terms of the bacterial community, Clostridia was the most abundant class in UR. Based on the results mentioned above, it is feasible to simultaneously improve both the CH₄ content and yield from rice straw by adding extra H₂ into the two-stage anaerobic digestion system.     

Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Biological hydrogen production: Simultaneous saccharification and fermentation with nitrogen and phosphorus removal from wastewater effluent

A simple anaerobic biodegradation process using wastewater treatment plant (WWTP) effluent, shredded paper, and a purge of nitrogen gas was used to produce hydrogen and simultaneously capture nitrogen and phosphorus. Two reactor configurations, a sequencing batch reactor (SBR) and a classic batch reactor (CBR) were tested as simultaneous saccharification and fermentation reactors (enzymatic hydrolysis and fermentation in one tank). The CBR demonstrated greater stability of hydrogen production and simplicity of operation, while the SBR provided better nitrogen and phosphorus removal efficiencies. Nuclear magnetic resonance analyses showed acetic acid to be the main product from both reactors. Optimal CBR conditions were found to be pH 5, 4 g/L loading, 0.45 ml/g Accellerase 1500, and 38 °C. Experiments with an argon purge in place of nitrogen and with ammonium chloride spiking suggested that hydrogenase and nitrogenase enzymes contributed similarly to hydrogen production in the cultures. Analysis of a single fermentation showed that hydrogen production occurred relatively early in the course of TOC removal, and that follow-on treatments might extract more energy from the products.     

Hydrogenase activity monitoring in the fermentative hydrogen production using heat pretreated sludge: A useful approach to evaluate bacterial communities performance

The conversion of organic compounds into H₂ has received increasing attention. Enrichment of inocula by heat pretreatment eliminates non-spore forming hydrogen consuming microorganisms and promotes spore germination in genus Clostridium, which is known as one of the key hydrogen producers. Useful information about metabolic pathway is provided by some intermediate metabolites, such as: acetic, propionic, butyric and formic acids. The increase of acetic/butyric acids ratio indicates H₂ production in heat pretreated inoculum when compared to untreated inoculum in the same cultivation conditions. The effect of heat pretreatment on inocula and consequently on the performance of bacterial communities responsible for H₂ production was monitored through the measurement of the level of hydrogenase gene expression, as well as through the content and distribution of volatile fatty acids. The acetic acid type fermentation was followed by the microorganisms presented in untreated and heat pretreated sludge. The medium containing untreated sludge presented a ratio of acetic/butyric acid of approximately 4, the same parameter was 7 when heat pretreated sludge was employed. The level of hydrogenase gene expression tripled when heat pretreated inoculum was used, indicating a higher production of H₂.     

Fermentative H2 production using a switchgrass steam exploded liquor fed to mixed anaerobic cultures: Effect of hydraulic retention time,linoleic acid and nitrogen sparging

Fermentative hydrogen (H₂) production from a steam exploded switchgrass liquor using inhibited mixed anaerobic microbial communities was studied in upflow anaerobic sludge blanket reactors (UASBRs). Increasing the H₂ yield was accomplished by treating the inoculum with linoleic acid (LA), varying the hydraulic retention time (HRT) and sparging liquid phase with nitrogen (N₂). A maximum H₂ yield of 2.56 ± 0.10 mol mol⁻¹ hexose, was obtained at a 6 h HRT in LA treated cultures sparged with N₂. Sparging or LA treatment alone was able to enhance the H₂ yield by 46 ± 5% and 38 ± 3%, respectively, in comparison to control cultures operating at a 6 h HRT. Of the different methods employed, N₂ sparging in combination with LA treatment proved to be more effective in enriching the H₂ producing bacteria belonging to Clostridium sp. Species belonging to Propionibacterium, Bacteroides and Eubacterium, which were associated with H₂ consumption and reduced byproducts formation, were observed in addition to Clostridium sp. in unsparged control cultures.