Molar Yield Research Articles

The role of anthraquinone-2-sulfonate on biohydrogen production by Klebsiella strain and mixed culture

The role of anthraquinone-2-sulfonate (AQS) on biohydrogen production was explored in this study. The hydrogen molar yield (HMY) of the batch experiment with 0.2 mM AQS was not significantly improved comparing to that without AQS, but the acetate and ethanol yields were increased by 12.1% and 9.82%, respectively. According to the metabolites flux analysis, e- balance calculations and stoichiometry results, the biogenic anthrahydroquinone-2,6,-disulfonate (AH2QS) preferred to improve the H2 generation that catalyzed by hydrogenase, but not likely affect the H2 generation that through formate cleavage pathway (FCP). However, comparing to the mix culture without AQS, the hydrogen production rate and hydrogen yield were increased by 20.97% of 1.96%, respectively, in the presence of AQS. The results of this study provided better understanding of the role of AQS on biohydrogen production by the strain that follows FCP, and the synergistic biohydrogen production by the co-culture that follows FCP and butyrate/acetate pathway.

The insights into the catalytic performance of rare earth metal ions on lactic acid formation from biomass via microwave heating

• Ln 3+ showed special activity for lactic acid formation from biomass. • The activity increased monotonically with the atomic number of Ln 3+ . • Smaller ionic radius of Ln 3+ led to stronger interaction with glucose. • Ln 3+ with smaller ionic radius exhibited higher electron-withdrawing ability. • The orbital-stabilization ability of transition state increased with atomic number. The unique properties of rare earth metal offer attractive potentials for the application as catalyst in biomass valorization. In present work, we found that rare earth metal ions exhibited specific catalytic activity for lactic acid production from biomass, giving 75.9% and 71.0% carbon molar yield of lactic acid from glucose and furfural residue, respectively. The activity of lanthanide(III) ions increased monotonically with the atomic number, as well as the decreasing ionic radius, from La 3+ to Lu 3+ . The increasing oxidation potential with atomic number might facilitate the disproportionation reaction in glucose conversion via an intramolecular H-shift. DFT calculation further revealed that smaller ionic radius of lanthanide(III) ions led to stronger interaction between lanthanide(III) ions and glucose/fructose, as well as the less composition of 4 f in LUMO that enabled more activation of glucose. More importantly, lanthanide(III) with smaller ionic radius exhibited higher electron-withdrawing ability from the oxygen atom in -C = O. This enhanced the electrophilicity of C2, C3, and C4 atoms, and led to the more exposure especially for C4 atom, thereby significantly weakening C3-C4 bond. In addition to the increasing orbital-stabilization ability at the bond-breaking site in the corresponding transition state with atomic number, the energy barrier for C3-C4 cleavage in fructose was greatly reduced by heavy lanthanide(III) ions. These factors contributed to the special activity for lactic acid formation from biomass. The insights in this work might give some useful clues for the widespread application of rare earth metal in biomass valorization.

Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for \u03b5-caprolactone production

ε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate

Catechol is an industrially relevant chemical with myriad applications. Its production via chemical route suffers from several drawbacks the major being a non-green and nonselective route. Currently, bio-based products using biocatalyst are gaining attention due to the growing environmental and health hazards concerns over the use of petroleum-derived feedstock. Lignocellulosic biomass serves as a promising feedstock. Lignin valorization is the demand of the current scenario which is complicated task by its complexity, heterogeneity and diversity of lignin structures posing limitations toward lignin valorization via chemical routes. There are several microorganisms that possess the ability to metabolize lignin monomers via their central metabolic pathways and this paves the way to the synthesis of a number of products. Pseudomonas putida KT2440 is one such organism and was chosen for genetic manipulations for catechol biosynthesis using lignin-derived model compounds and biomass hydrolysate stream comprising of various lignin monomers. Catechol production was engineered by diverting various lignin monomers and addressing the identified metabolic bottlenecks particularly vanillic acid accumulation toward catechol biosynthesis. The engineered strain could convert the model lignin monomers as well as monomers in the biomass hydrolysates to catechol and vanillic acid in more than 60% and 90% molar yields, respectively.

Large contribution to secondary organic aerosol from isoprene cloud chemistry.

Aerosols still present the largest uncertainty in estimating anthropogenic radiative forcing. Cloud processing is potentially important for secondary organic aerosol (SOA) formation, a major aerosol component: however, laboratory experiments fail to mimic this process under atmospherically relevant conditions. We developed a wetted-wall flow reactor to simulate aqueous-phase processing of isoprene oxidation products (iOP) in cloud droplets. We find that 50 to 70% (in moles) of iOP partition into the aqueous cloud phase, where they rapidly react with OH radicals, producing SOA with a molar yield of 0.45 after cloud droplet evaporation. Integrating our experimental results into a global model, we show that clouds effectively boost the amount of SOA. We conclude that, on a global scale, cloud processing of iOP produces 6.9 Tg of SOA per year or approximately 20% of the total biogenic SOA burden and is the main source of SOA in the mid-troposphere (4 to 6 km).

Streptomyces tunisiensis DSM 42037 mediated bioconversion of ferulic acid released from barley bran.

Streptomyces tunisiensis DSM 42037 exhibited growth capacity on a minimum medium containing 1% barley bran. This peculiar strain released 83.5% of total ferulic acid present in barley bran after 5days of incubation and the highest amount of released ferulic acid (19mg/L) was observed on the 3rd day of incubation. The concentrated supernatant of S. tunisiensis also released ferulic acid from the parietal arabinoxylan complex of barley bran. This strain was able to convert the free ferulic acid into 4-vinyl guaiacol (14mg/L) and acetovanillone (12mg/L) at molar yield of 97% and 83% respectively. The biotransformation products were successively purified by preparative thin layer and silica gel column chromatography followed by HPLC and identified by 1H nuclear magnetic resonance. Streptomyces tunisiensis DSM 42037 could have potential applications in the food, pharmaceutical and cosmetic industries thanks to its ability in biotransforming ferulic acid into 4-vinyl guaiacol and acetovanillone.

Gas-phase ozonolysis of trans-2-hexenal: Kinetics, products, mechanism and SOA formation

In this work, kinetics, product formation, chemical mechanism and SOA formation for the gas-phase reaction of trans-2-hexenal (T2H) with O3 are examined using four complementary experimental setups at 298 ± 2 K and atmospheric pressure. Product studies were conducted in two contrasted experimental conditions, with and without OH radical scavenger. The ozonolysis rate constant was determined in both static and dynamic reactors. An average reaction rate constant of (1.52 ± 0.19) × 10−18 cm3 molecule−1 s−1 was determined. Glyoxal and butanal were identified as main products with molar yields of 59 ± 15% and 36 ± 9%, respectively, in the presence of an OH scavenger. Slightly lower values were obtained in the absence of scavenger. Acetaldehyde, propanal and 2-hydroxybutanal were also identified and quantified. A reaction mechanism was proposed based on the observed products. SOA formation was observed with aerosol mass yields >13% for SOA masses of 400 μg m−3. This work demonstrates for the first time that 2-alkenals ozonolysis can be a source of SOA in the atmosphere.

Gas-Particle Partitioning and SOA Yields of Organonitrate Products from NO3-Initiated Oxidation of Isoprene under Varied Chemical Regimes.

Alkyl nitrate (AN) and secondary organic aerosol (SOA) from the reaction of nitrate radicals (NO3) with isoprene were observed in the Simulation of Atmospheric PHotochemistry In a large Reaction (SAPHIR) chamber during the NO3Isop campaign in August 2018. Based on 15 day-long experiments under various reaction conditions, we conclude that the reaction has a nominally unity molar AN yield (observed range 90 ± 40%) and an SOA mass yield of OA + organic nitrate aerosol of 13–15% (with ∼50 μg m–3 inorganic seed aerosol and 2–5 μg m–3 total organic aerosol). Isoprene (5–25 ppb) and oxidant (typically ∼100 ppb O3 and 5–25 ppb NO2) concentrations and aerosol composition (inorganic and organic coating) were varied while remaining close to ambient conditions, producing similar AN and SOA yields under all regimes. We observe the formation of dinitrates upon oxidation of the second double bond only once the isoprene precursor is fully consumed. We determine the bulk partitioning coefficient for ANs (Kp ∼ 10–3 m3 μg–1), indicating an average volatility corresponding to a C5 hydroxy hydroperoxy nitrate.

An experimental study of the gas-phase reaction between Cl atoms and trans-2-pentenal: Kinetics, products and SOA formation

The gas-phase reaction of trans-2-pentenal (T2P) with Cl atoms was studied at atmospheric pressure and room temperature. A rate coefficient of (2.56 ± 0.83) × 10-10 cm3 molecule-1 s-1 was obtained using the relative rate method and isoprene, cyclohexane and ethanol as reference compounds. The kinetic study was carried out using a 300-L Teflon bag simulation chamber (IMT Lille Douai-France) and a 16-L Pyrex cell (UCLM-Ciudad Real-Spain), both coupled to the Fourier transform infrared (FTIR) technique. Gas-phase products and secondary organic aerosol (SOA) formation were studied at UCLM using a 16-L Pyrex cell and a 264-L quartz simulation chamber coupled to the FTIR and gas-chromatography-mass spectrometry (GC-MS) techniques. HCl, CO, and propanal were identified as products formed from the studied reaction and quantified by FTIR, the molar yield of the latter being (5.2 ± 0.2)%. Formic acid was identified as a secondary product and was quantified by FTIR with a yield of (6.2 ± 0.4)%. In addition, 2-chlorobutanal and 2–pentenoic acid were identified, but not quantified, by GC-MS as products. The SOA formation was investigated using a fast mobility particle sizer spectrometer. The observed SOA yields reached maximum values of around 7% at high particle mass concentrations. This work provides the first study of the formation of gaseous and particulate products for the reaction of Cl with T2P. A reaction mechanism is suggested to explain the formation of the observed gaseous products. The results are discussed in terms of structure-reactivity relationship, and the atmospheric implications derived from this study are commented as well.

Biodegradation of lignin monomers and bioconversion of ferulic acid to vanillic acid by Paraburkholderia aromaticivorans AR20-38 isolated from Alpine forest soil

Lignin bio-valorization is an emerging field of applied biotechnology and has not yet been studied at low temperatures. Paraburkholderia aromaticivorans AR20-38 was examined for its potential to degrade six selected lignin monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, vanillic acid, benzoic acid) from different upper funneling aromatic pathways. The strain degraded four of these compounds at 10°C, 20°C, and 30°C; syringic acid and vanillic acid were not utilized as sole carbon source. The degradation of 5 mM and 10 mM ferulic acid was accompanied by the stable accumulation of high amounts of the value-added product vanillic acid (85–89% molar yield; 760 and 1540 mg l−1, respectively) over the whole temperature range tested. The presence of essential genes required for reactions in the upper funneling pathways was confirmed in the genome. This is the first report on biodegradation of lignin monomers and the stable vanillic acid production at low and moderate temperatures by P. aromaticivorans.Key points• Paraburkholderia aromaticivorans AR20-38 successfully degrades four lignin monomers.• Successful degradation study at low (10°C) and moderate temperatures (20–30°C).• Biotechnological value: high yield of vanillic acid produced from ferulic acid.

Optimizing a Fed-Batch High-Density Fermentation Process for Medium Chain-Length Poly(3-Hydroxyalkanoates) in Escherichia coli.

Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by β-oxidation deficient recombinant Escherichia coli LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L–1). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L–1, 0.42 g L–1 h–1), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L–1). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.

Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli.

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.

Development of Pseudomonas asiatica as a host for the production of 3-hydroxypropionic acid from glycerol

Pseudomonas asiatica C1, which could grow on glucose and aerobically synthesize coenzyme B12, was isolated and developed as a microbial cell factory for the production of 3-hydroxypropionic acid (3-HP) from glycerol. Three heterologous enzymes, glycerol dehydratase (GDHt), GDHt reactivase (GdrAB) and aldehyde dehydrogenase (ALDH), constituting the 3-HP synthesis pathway, were introduced, and three putative dehydrogenases, responsible for 3-HP degradation, were disrupted. In addition, the transcriptional repressor glpR and the glycerol kinase glpK were removed to increase glycerol import while eliminating the catabolic use of glycerol. Furthermore, the global regulatory protein encoded by crc and several putative oxidoreductases (PDORs) were disrupted. One resulting strain, when grown on glucose, could produce 3-HP at ~ 700 mM in 48 h in a fed-batch bioreactor experiment, with the molar yield > 0.99 on glycerol without much by-products. This study demonstrates that P. asiatica C1 is a promising host for production of 3-HP from glycerol.

Preparation of minor ginsenosides C-K and C-Mx from protopanaxadiol ginsenosides of American ginseng leaves by a enzyme from Aspergillus sp.agl-84 strain

To improve utilization of American-ginseng-leaf ginsenosides, the PPD-ginsenosides from American-ginseng-leaves was reacted by a ginsenosidase type-I from Aspergillus sp.agl-84 to prepare minor ginsenosides C-K and C-Mx. PPD-ginsenosides, containing over 79 % of Rb3 and Rd, were separated from American-ginseng-leaves; the yield was 10.5 %. The molecular weight of ginsenosidase type-I from Aspergillus sp.agl-84 was approximately 75.2 kDa; Michaelis constants Km and Vmax were 14.8 mM and 58.6 mM/h for Rb3, 15.4 mM and 56.8 mM/h for Rd. The enzyme reaction velocities on 3-O-glucosides of Rb3 and Rd were higher than that of other Aspergillus strain ginsenosidase type-I to prove that the enzyme was more suitable for conversion of PPD-ginsenosides from American-ginseng-leaves into C-K and C-Mx. According to enzyme reaction dynamic, 2.5 % PPD-ginsenosides were reacted at 45 °C and pH 5.0 for 24 h by a crude ginsenosidase type-I from Aspergillus sp.agl-84 strain. Here, 4.1 g C-K and 4. 2 g C-Mx were obtained from 20 g PPD-ginsenosides separating from 190 g American-ginseng-leaves. C-K molar yield was 34.3 %, C-Mx molar yield was 29.0 %, the total molar yield was 63.3 %. Therefore, minor ginsenosides C-K and C-Mx were successfully prepared from PPD-ginsenosides of American-ginseng-leaves using crude enzyme from Aspergillus sp.agl-84 at low cost.

Understanding the Yields of Giant Unilamellar Vesicles Composed of Charged Membranes

Double-layer electrostatic interactions play an important role in colloid and surface phenomena including for biological membranes. While important for recapitulating biophysical phenomena such as virus fusion, protein binding, and for tuning interactions with biomedically important surfaces, the role of membrane electrostatic interactions in the surface-assisted assembly of cell-like giant unilamellar vesicles, GUVs, is still poorly understood. Here, we show that both the molar yield and the sizes of GUVs falls systematically with increasing mol fraction of the anionic charged lipids DOPG and DOPS. We use the budding and merging (BNM) model for the assembly of GUVs to understand the effect of electrostatic interactions. In the BNM model, GUVs assemble through the budding of nanoscale buds which then merge to form micrometer scale buds. Our calculations show that although membranes with higher mol fraction of charged lipids experience decreased interbilayer adhesive interactions thus reducing the energy cost for budding, the increased electrostatic potential between the nanoscale buds raises the energy cost for merging. The higher barrier to merging explains our result that increasing the fraction of charged lipids in the membrane decreases GUV yields and sizes. It also explains previous observations in the literature that it is difficult to obtain GUVs with membranes composed of anionic lipids. We show that the higher barrier to merging can be overcome by crowding the surface with nanoscale buds. We can obtain high yields of GUVs with charged membranes by increasing the surface concentration of lipids. These results enable us to optimize the yield of lipid mixtures that reflect physiological compositions of cell membranes and open up avenues into larger-scale production for applications that require cell mimics.

Oxidants Emergence under Dual-Frequency Sonication within Single Acoustic Bubble: Effects of Frequency Combinations

The sonochemical activity of an oxygen bubble formed in an aqueous medium and oscillating under a dual-frequency excitation is studied in this paper. Several couples of frequencies are formed amongst 35, 140, 300, and 515 kHz, with a maximum acoustic amplitude of 1.5 atm. The molar yields of the emerging oxidants are analyzed in accordance with the combined frequencies and compared to the cases of mono-frequency excitations of similar maximum amplitude, i.e., 1.5 atm. Qualitatively, passing from a mono to a dual-frequency excitation demonstrated to be with no effect on the predominant species which remain, and. However, the results exhibited a very selective quantitative evolution depending on the combined frequencies. Some couples proved to induce a negative effect and reduce the production at the single bubble level, particularly with basic frequencies of 140 and 300 kHz, while some others demonstrated a noticeable enhancement such as the couples (35, 140 kHz) and (515, 35 kHz), as compared to mono-frequency fields.

Thermodynamic modelling of dimethyl ether steam reforming

Hydrogen is a promising clean energy strategy that can be obtained from the steam reforming of hydrogen-rich materials such as dimethyl ether (DME). In this study, the results of thermodynamic analysis of DME steam reforming were utilised as a framework for the development of correlations for predicting percentage molar yield of each species given a known set of inputs. Response surface methodology—historical data design was used for the work. The effect of temperature, pressure and steam to carbon ratio was shown to be significant in the process. Combinatorial effects of factors and the yield of various chemical species in the product stream were elucidated. The key contribution of this paper is the development of model predictors of all chemical species in the steam reforming of DME. All models developed were shown to be significant. These can afford for quick predictions given a known set of process inputs and can play an important role in the design of DME reforming systems.

Evaluation of Histidine Reactivity and Byproduct Formation during Peptide Chlorination

The covalent modifications resulting from chlorine reactions with peptide-bound amino acids contribute to pathogen inactivation and disinfection byproduct (DBP) formation. Previous research suggested that histidine is the third most reactive of the seven chlorine-reactive amino acids, leading to the formation of 2-chlorohistidine, 2-oxohistidine, or low-molecular-weight byproducts such as trihalomethanes. This study demonstrates that histidine is less reactive toward formation of chlorine transformation products (transformation time scale of hours to days) than five of the seven chlorine-reactive amino acids, including tyrosine (transformation time scale of minutes). Chlorine targeted tyrosine in preference to histidine within peptides, indicating that chlorine reactions with tyrosine and other more reactive amino acids could contribute more to the structural modifications to proteins over the short time scales relevant to pathogen inactivation. Over the longer time scales relevant to disinfection byproduct formation in treatment plants or distribution systems, this study identified β-cyanoalanine as the dominant transformation product of chlorine reactions with peptide-bound histidine, with molar yields of ∼50% after 1 day. While a chlorinated histidine intermediate was observed at lower yields (maximum ∼5%), the cumulative concentration of the conventional low-molecular-weight DBPs (e.g., trihalomethanes) was ≤7%. These findings support the need to identify the high-yield initial transformation products of chlorine reactions with important precursor structures to facilitate the identification of unknown DBPs.

Cascade biocatalysis for production of enantiopure (S)-2-hydroxybutyric acid using recombinant Escherichia coli with a tunable multi-enzyme-coordinate expression system

Racemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application. Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) for producing optically pure (S)-2-hydroxybutyric acid ((S)-2-HBA) from bulk chemical L-threonine. To solve the mismatch in the conversion rate and the consumption rate of intermediate 2-oxobutyric acid (2-OBA) formed in the multi-enzyme catalysis reaction, ribosome binding site regulation strategy was explored to control TD expression levels, achieving an eightfold alteration in the conversion rate of 2-OBA. With the optimized activity ratio of the three enzymes and using ADH for NADH regeneration, the recombinant strain ADH-r53 showed increased production of (S)-2-HBA with the highest titer of 129 g/L and molar yield of 93% within 24 h, which is approximately 1.65 times that of the highest yield reported so far. Moreover, (S)-2-HBA could easily be purified by distillation, making it have great potential for industrial application. Additionally, our results indicated that constructing a tunable multi-enzyme-coordinate expression system in single cell had great significance in biocatalysis of hydroxyl acids.

Hydrogen Production by Immobilized Cells of Clostridium intestinale Strain URNW Using Alginate Beads.

Biological hydrogen (H2) is a promising candidate for production of renewable hydrogen. Using entrapped cells rather than conventional suspended cell cultures for the production of H2 offers several advantages, such as improved production yields related to higher cell density, and enhanced resistance to substrate and end-product inhibition. In this study, H2 production by a novel isolate of Clostridium intestinale (strain URNW) was evaluated using cells entrapped within 2% calcium-alginate beads under strictly anaerobic conditions. Both immobilized cells and suspended cultures were studied in sequential batch-mode anaerobic fermentation over 192 h. The production of H2 in the headspace was examined for four different initial cellobiose concentrations (5, 10, 20, and 40 mM). Although a lag period for initiation of the fermentation process was observed for bacteria entrapped within hydrogel beads, the immobilized cells achieved both higher volumetric production rates (mmol H2/(L culture h)) and molar yields (mol H2/mol glucose equivalent) of H2 compared with suspended cultures. In the current study, the maximum cellobiose consumption rate of 0.40 mM/h, corresponding to 133.3 mg/(L h), was achieved after 72 h of fermentation by immobilized cells, generating a high hydrogen yield of 3.57 mol H2/mol cellobiose, whereas suspended cultures only yielded 1.77 mol H2/mol cellobiose. The results suggest that cells remain viable within the hydrogels and proliferated with a slow rate over the course of fermentation. The stable productivity of immobilized cells over 8 days with four changes of medium depicted that the immobilized cells of the isolated strain can successfully yield higher hydrogen and lower soluble metabolites than suspended cells suggesting a feasible process for future applications for bioH2 production.