Major Soluble Metabolites Research Articles

Isolation and Characterization of Biohydrogen-Producing Bacteria for Biohydrogen Fermentation Using Oil Palm Biomass-Based Carbon Source

The effectiveness of biohydrogen conversion from biomass sources is governed by the selection of ideal biohydrogen-producing bacteria to achieve high and consistent production performance. The aim of this research was to isolate and identify a biohydrogen producer in local soil samples, as well as to evaluate its fermentability in biohydrogen production from oil palm empty fruit bunches (OPEFB). To this end, preliminary identification was performed using morphological, phenotype, biological, and 16s rRNA analyses. The fermentability of the isolate was further evaluated in a serum bottle and then in a 1.5 L anaerobic column bioreactor (ACBR) to investigate the potential for biohydrogen production using two OPEFB-based carbon sources: hydrolysate of ammonia fiber expansion (AFEX)-pretreated OPEFB and molasses from dilute acetic acid (DAA)-pretreated OPEFB. The isolated strain, Enterobacter sp. KBH 6958, was found to be capable of producing biohydrogen from various carbon sources via the pyruvate:ferredoxin oxidoreductase (PFOR) pathway. The cumulative conversion of AFEX OPEFB hydrolysate was 45% higher than that observed in DAA OPEFB molasses fermentation in the production of biohydrogen. The biohydrogen yield after fermenting AFEX OPEFB hydrolysate with Enterobacter sp. KBH 6958 was 1.55 mol H2/mol sugar, with a maximum productivity of 98.1 mL H2/h (4.01 mmol H2/L/h), whereas butyrate (10.6 mM), acetate (11.8 mM), and ethanol (4.56 mM) were found to be the major soluble metabolites. This study successfully demonstrated the biotechnological conversion of OPEFB into biohydrogen using a locally isolated strain, which not only solves environmental issues associated with the industry but may also offer a solution to the world’s energy insecurity.

Dark fermentation of complex waste biomass for biohydrogen production by pretreated thermophilic anaerobic digestate

The Biohydrogen Potential (BHP) of six different types of waste biomass typical for the Campania Region (Italy) was investigated. Anaerobic sludge pre-treated with the specific methanogenic inhibitor sodium 2-bromoethanesulfonic acid (BESA) was used as seed inoculum. The BESA pre-treatment yielded the highest BHP in BHP tests carried out with pre-treated anaerobic sludge using potato and pumpkin waste as the substrates, in comparison with aeration or heat shock pre-treatment. The BHP tests carried out with different complex waste biomass showed average BHP values in a decreasing order from potato and pumpkin wastes (171.1 ± 7.3 ml H2/g VS) to buffalo manure (135.6 ± 4.1 ml H2/g VS), dried blood (slaughter house waste, 87.6 ± 4.1 ml H2/g VS), fennel waste (58.1 ± 29.8 ml H2/g VS), olive pomace (54.9 ± 5.4 ml H2/g VS) and olive mill wastewater (46.0 ± 15.6 ml H2/g VS). The digestate was analyzed for major soluble metabolites to elucidate the different biochemical pathways in the BHP tests. These showed the H2 was produced via mixed type fermentation pathways.

Characteristics and kinetics of biohydrogen production with Ni2+ using hydrogen-producing bacteria

The characteristics and kinetics of biohydrogen production with Ni2+ using hydrogen-producing bacteria (HPB) obtained from cow dung were investigated in batch tests. The results indicated that cumulative biohydrogen production and hydrogen yield were enhanced with an increase of 0.3–6 mg/L in the Ni2+ concentration at 35 °C and an initial pH of 6.0. The maximum cumulative biohydrogen production was 442.52 mL, and the maximum hydrogen yield was 2.05 mol H2/mol sucrose at 0.6 mg/L Ni2+. Compared with the control, the cumulative biohydrogen production with 0.6 mg/L Ni2+ was doubled. The major soluble metabolites produced in this study were ethanol, acetate, and butyrate. In addition, the final pH was lower than the initial pH after the production of biohydrogen, and the substrate degradation efficiency was slightly enhanced with an increase in the Ni2+ concentration from 0.3 to 9 mg/L. Modified Gompertz and Han–Levenspiel models were used to describe the effects of Ni2+ on biohydrogen production, and the model predicted data fit the experimental results well. The mathematical constants n and m of these models were 103.59 and 2.69, respectively, suggesting that the reaction was uninhibited. The predicted critical Ni2+ concentration was 15.58 mg/L, which completely suppressed biohydrogen production. This study contributes to the improvement of organic waste utilisation by HPB.

음폐수 소화액에 포함된 acetate가 Chlorella vulgaris의 성장 및 질소, 인 제거에 미치는 영향

VFAs like acetate are the major soluble metabolites of food waste leachates after digested. Therefore this study investigates the effect of acetate on growth rate and nutrient removal efficiency of Chlorella vulgaris to treat digested food waste leachates. The initial acetate concentration varied from 0 to 20 mM. As a result, Chlorella vulgaris growth rate was increased as high as the concentrations ranged from 0 to 20 mM. The same trend was observed with -N and -P consumption. The highest growth rate and the highest -N, -P removal rate were observed at acetate concentration of 20 mM. The microalgae growth rate and -N, -P removal rates were 1.5, 1.8, 2.3 times higher than the condition without acetate.

Biohydrogen Production from Textile Wastewater by Mixed Microflora in an Intermittent‐flow, Stirred Tank Reactor: Effect of Feeding Frequency

AbstractAn intermittent‐flow, stirred tank reactor (IFSTR) was used to produce biohydrogen from textile wastewater (TW) by activated sludge. The IFSTR was operated at pH 5.5 under various feeding frequencies and TW concentrations. The first intermittent peak biogas production rate of 17.5 L biogas/L‐d and HPR of 10 L H2/L‐d were obtained at feeding frequency of 12 times/d with TW concentration of 33.1 g hexoses/L. Butyrate and acetate were the major soluble metabolites at peak HPR, whereas lactate and ethanol accumulation decreased the HPR. Close resemblance of bacterial communities in periods of peak HPR with hydrogen producer Clostridium butyricum was observed.

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H2/mol glucose, 2.2 ± 0.2 mol H2/mol xylose and 5.2 ± 0.2 mol H2/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.

Batch fermentative hydrogen production by enriched mixed culture: Combination strategy and their microbial composition

The effect of individual and combined mixed culture on dark fermentative hydrogen production performance was investigated. Mixed cultures from cow dung (C1), sewage sludge (C2), and pig slurry (C3) were enriched under strict anaerobic conditions at 37°C with glucose as the sole carbon source. Biochemical hydrogen production test in peptone-yeast-glucose (PYG) and basal medium was performed for individual mixed cultures (C1, C2 and C3) and their combinations (C1-C2, C2-C3, C1-C3 and C1-C2-C3) at a glucose concentration of 10 g/L, 37°C and initial pH 7. Maximum hydrogen yields (HY) of 2.0 and 1.86 [Formula: see text] by C2, and 1.98 and 1.95 mol(H2)/mol(glucose) by C2-C3 were obtained in PYG and basal medium, respectively. Butyrate and acetate were the major soluble metabolites produced by all the cultures, and the ratio of butyrate to acetate was ∼2 fold higher in basal medium than PYG medium, indicating strong influence of media formulation on glucose catabolism. The major hydrogen-producing bacterial strains, observed in all mixed cultures, belonged to Clostridium butyricum, C. saccharobutylicum, C. tertium and C. perfringens. The hydrogen production performance of the combined mixed culture (C2-C3) was further evaluated on beverage wastewater (10 g/L) at pH 7 and 37°C. The results showed an HY of 1.92 mol(H2)/mol(glucose-equivalent). Experimental evidence suggests that hydrogen fermentation by mixed culture combination could be a novel strategy to improve the HY from industrial wastewater.

Effect of furans and linoleic acid on hydrogen production

The effects of furans (furfural and 5-hydroxymethylfurfural (HMF)) on hydrogen (H2) production using mixed anaerobic cultures were evaluated by conducting batch experiments. Two mixed anaerobic cultures (culture A and B) fed furans plus glucose and treated with and without linoleic acid (LA) at pH 5.5 were maintained at 37 °C. In the LA inhibited cultures A and B fed 0.75 g L−1 furfural and 0.25 g L−1 HMF, the maximum H2 yields observed were 1.89 ± 0.27 mol mol−1 glucose and 1.75 ± 0.22 mol mol−1 glucose, respectively. In cultures with maximum H2 yields, Clostridium sp. and Flavobacterium sp. were dominant. Acetate, butyrate and ethanol were the major soluble metabolites detected in cultures A and B whereas propionate was also dominant in culture B. A canonical correspondence analysis based on the byproducts and the relative abundance of the terminal-restriction fragments revealed less variation between cultures treated with LA and low correlation value between the factors and the species composition.

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C5H10O5). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H2/mol pentose and the highest productivity rate achieved was 0.91 mmol H2/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H2/mol pentose while the maximum productivity was 1.1 mmol H2/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated UASB granules as affected by organic loading rate

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated upflow anaerobic sludge blanket (UASB) granules was examined in a UASB reactor. The organic loading rate (OLR) was optimized in order to maximize the hydrogen production rate (HPR). The maximum hydrogen content (37.1% and 24.2%) and HPR (9 and 6.2 mmol H2/L h) were achieved at the optimum OLR of 50 g/L d using pure and waste glycerol as the substrate, respectively. The major soluble metabolite products (SMPs) were ethanol, 1,3-propanediol (1,3-PD), formic acid, and acetic acid. The microbial community and microbial structure, analyzed by fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM), revealed that the predominant hydrogen producers were E. aerogenes ATCC 13048 and firmicutes bacteria including Clostridium, Bacillus, and Dialister sp.

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

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

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.

Application of polyethylene glycol immobilized Clostridium sp. LS2 for continuous hydrogen production from palm oil mill effluent in upflow anaerobic sludge blanket reactor

A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium sp. LS2 for continuous hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. Palm oil mill effluent (POME) was used as the substrate carbon source. The optimal amount of PEG-immobilized cells for anaerobic hydrogen production was 12% (w/v) in the UASB reactor. The UASB reactor containing immobilized cells was operated at varying hydraulic retention times (HRT) that ranged from 24 to 6h at 3.3g chemical oxygen demand (COD)/L/h organic loading rate (OLR), or at OLRs that ranged from 1.6 to 6.6 at 12h HRT. The best volumetric hydrogen production rate of 336mL H2/L/h (or 15.0mmol/L/h) with a hydrogen yield of 0.35L H2/g CODremoved was obtained at a HRT of 12h and an OLR of 5.0g COD/L/h. The average hydrogen content of biogas and COD reduction were 52% and 62%, respectively. The major soluble metabolites during hydrogen fermentation were butyric acid followed by acetic acid. It is concluded that the PEG-immobilized cell system developed in this work has great potential for continuous hydrogen production from real wastewater (POME) using the UASB reactor.

Preclinical Evaluation of the Metabolism and Disposition of RRx-001, a Novel Investigative Anticancer Agent

RRx-001 has shown promise as a novel cancer therapeutic agent. The disposition of RRx-001 was evaluated in vitro and after intravenous administration to rats. At both 24 and 168 h after a single intravenous administration of ¹⁴C-RRx-001 (10 mg/kg), the majority of radiolabel was in the blood. The recovery of label in excreta was quite low, but the major route of radiolabel excretion was via the kidney, with approximately 26% in the urine by the first 8 h and decreasing amounts in all subsequent collections to a total of 36.3% by 168 h. The partitioning of total radioactivity in red blood cells (RBCs) and plasma was determined after in vitro addition to human, rat, dog, and monkey whole blood at 1 and 20 μM. In rat, at 30 min, approximately 75% of the radioactivity is associated with RBCs and 25% with plasma. In human, at 30 min, approximately 25% of the radioactivity is associated with RBCs and 75% with plasma. Analysis by liquid chromatography/radiodetection/mass spectrometry showed that ¹⁴C-RRx-001 reacted rapidly with whole blood to give four major soluble metabolites: the GSH and Cys adducts of RRx-001 (M1 and M2) and the corresponding mononitro GSH and Cys adducts (M3 and M4). Human Hb was incubated with cold RRx-001 in buffer, and a standard proteomics protocol was used to separate and identify the tryptic peptides. Standard peptide collision-induced fragment ions supported the structure of the peptide GTFATLSELHCDK with the alkylation on the Cys-93 locus of the Hb β chain.

Mesophilic fermentative hydrogen production from sago starch-processing wastewater using enriched mixed cultures

Biohydrogen (bioH2) production from starch-containing wastewater is an energy intensive process as it involves thermophilic temperatures for hydrolysis prior to dark fermentation. Here we report a low energy consumption bioH2 production process with sago starch powder and wastewater at 30 °C using enriched anaerobic mixed cultures. The effect of various inoculum pretreatment methods like heat (80 °C, 2 h), acid (pH 4, 2.5 N HCl, 24 h) and chemical (0.2 g L−1 bromoethanesulphonic acid, 24 h) on bioH2 production from starch powder (1% w/v) showed highest yield (323.4 mL g−1 starch) in heat-treatment and peak production rate (144.5 mL L−1 h−1) in acid-treatment. Acetate (1.07 g L−1) and butyrate (1.21 g L−1) were major soluble metabolites of heat-treatment. Heat-treated inoculum was used to develop mixed cultures on sago starch (1% w/v) in minimal medium with 0.1% peptone-yeast extract (PY) at initial pH 7 and 30 °C. The effect of sago starch concentration, pH, inoculum size and nutrients (PY and Fe ions) on batch bioH2 production showed 0.5% substrate, pH 7, 10% inoculum size and 0.1% PY as the best H2 yielding conditions. Peak H2 yield and production rate were 412.6 mL g−1 starch and 78.6 mL L−1 h−1, respectively at the optimal conditions. Batch experiment results using sago-processing wastewater under similar conditions showed bioH2 yield of 126.5 mL g−1 COD and 456 mL g−1 starch. The net energy was calculated to be +2.97 kJ g−1 COD and +0.57 kJ g−1 COD for sago starch powder and wastewater, respectively. Finally, the estimated net energy value of +2.85 × 1013 kJ from worldwide sago-processing wastewater production indicates that this wastewater can serve as a promising feedstock for bioH2 production with low energy input.

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H2/L substrate·d and 1.34 mol H2/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H2/L substrate·d and the highest HY of 1.52 mol H2/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.

Temperature stress effects in Quercus suber leaf metabolism

Based on projections that climate changes are will intensify in the near future, it is important to understand how plants respond to climate. Consequently, we have been studying the effect of contrasting temperatures on leaf metabolism of Quercus suber, an important Mediterranean oak. Potted plants were grown under controlled conditions for 53 days at 28°C or 10°C. The accumulation of major soluble metabolites was analyzed by NMR. The relative levels of transcripts of genes encoding key enzymes of the shikimate and phenylpropanoid pathway (CS, PAL, CAD and ChS) were examined by means of quantitative, real-time RT-PCR. At 10°C, in the pre-existing leaves, the concentrations of sucrose, quercitol and catechin were higher, as were PAL and ChS transcripts. At 28°C, however, it was the concentration of quinic acid that was higher, as were the concentrations of CS and CAD transcripts. We conclude that contrasting temperatures greatly influence Q. suber metabolism and that a deeper analysis of the effects of more extreme temperatures is needed to understand the possible effects of temperature changes on Q. suber metabolism and physiology.

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H 2/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.

Anaerobic fluidized bed reactor with expanded clay as support for hydrogen production through dark fermentation of glucose

This study evaluated hydrogen production in an anaerobic fluidized bed reactor (AFBR) fed with glucose-based synthetic wastewater. Particles of expanded clay (2.8–3.35 mm) were used as a support material for biomass immobilization. The reactor was operated with hydraulic retention times (HRT) ranging from 8 to 1 h. The hydrogen yield production increased from 1.41 to 2.49 mol H 2 mol −1 glucose as HRT decreased from 8 to 2 h. However, when HRT was 1 h, there was a slight decrease to 2.41 mol H 2 mol −1 glucose. The biogas produced was composed of H 2 and CO 2, and the H 2 content increased from 8% to 35% as HRT decreased. The major soluble metabolites during H 2 fermentation were acetic acid (HAc) and butyric acid (HBu), accounting for 36.1–53.3% and 37.7–44.9% of total soluble metabolites, respectively. Overall, the results demonstrate the potential of using expanded clay as support material for hydrogen production in AFBRs.

Influence of Ni 2+ concentration on biohydrogen production

In this paper, the effect of Ni 2+ concentration ranging from 0 to 50 mg/L on fermentative hydrogen production by mixed cultures was investigated in batch test. The results showed that at 35 °C and initial pH 7.0, Ni 2+ was able to enhance the hydrogen production rate with increasing Ni 2+ concentration from 0 to 0.2 mg/L, and enhance the hydrogen production potential and hydrogen yield with increasing Ni 2+ concentration from 0 to 0.1 mg/L. The maximum hydrogen production potential of 288.6 mL and the maximum hydrogen yield of 296.1 mL/g glucose were obtained at the Ni 2+ concentration of 0.1 mg/L. In all tests, the major soluble metabolites produced by mixed cultures were ethanol, acetic acid and butyric acid, without propionic acid. Ni 2+ had little effect on the substrate degradation efficiency with increasing concentration from 0 to 50 mg/L. Ni 2+ was able to enhance the biomass production yield with increasing Ni 2+ concentration from 0 to 0.1 mg/L. The maximum biomass production yield of 232.5 mg/g glucose was obtained at the Ni 2+ concentration of 0.1 mg/L. In all tests, the final pH after fermentative hydrogen production was lower than the initial pH.