Performance characteristics of a two-stage dark fermentative system producing hydrogen and methane continuously

The performance of a mesophilic two-stage system generating hydrogen and methane continuously from sucrose (10-30 g/L) was investigated. A hydrogen-generating CSTR followed by an upflow anaerobic filter were both inoculated with anaerobically digested sewage sludge, and ORP, pH, gas output, %H(2), %CH(4) and %CO(2) monitored. pH was controlled with NaOH, KOH or Ca(OH)(2). Using NaOH as alkali with 10 g/L sucrose, yields of 1.62 +/- 0.2 mol H(2)/mol hexose added and 323 mL CH(4)/gCOD added to the hydrogen and methane reactors respectively were achieved. The overall chemical oxygen demand (COD) reduction was 92.6% with 0.90 +/- 0.1 g/L sodium and 316 +/- 40 mg/L residual acetate in the methane reactor. Operation at 20 g/L sucrose and NaOH as alkali led to impaired volatile fatty acid (VFA) degradation in the methane reactor with 2.23 +/- 0.2 g/L sodium, 1,885 mg/L residual acetate, a hydrogen yield of 1.47 +/- 0.1 mol/mol hexose added, a methane yield of 294 mL/gCOD added and an overall COD reduction of 83%. Using Ca(OH)(2) as alkali with 20 g/L sucrose gave a hydrogen yield of 1.29 +/- 0.3 mol/mol hexose added, a methane yield of 337 mL/gCOD added and improved the overall COD reduction to 91% with residual acetate concentrations of 522 +/- 87 mg/L. Operation at 30 g/L sucrose with Ca(OH)(2) gave poorer overall COD reduction (68%), a hydrogen yield of 1.47 +/- 0.2 mol/mol hexose added, a methane yield of 138 mL/gCOD added and residual acetate 7,343 +/- 715 mg/L. It was shown that sodium toxicity and overloading are important issues for successful anaerobic digestion of effluent from biohydrogen reactors in high rate systems.     

Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora

For the transition to the hydrogen economy, hydrogen must be produced sustainably, e.g., by the fermentation of agricultural material. Continuous fermentative production of hydrogen from an insoluble substrate in nonsterile conditions is yet to be reported. In this study hydrogen production using mixed microflora from heat-treated digested sewage sludge in nonsterile conditions from a particulate co-product of the wheat flour industry (7.5 g L(-1) total hexose) at 18- and 12-hour hydraulic retention times, pH 4.5 and 5.2, 30 degrees C and 35 degrees C was examined. In continuous operation, hydrogen yields of approximately 1.3 moles hydrogen/mole hexose consumed were obtained, but decreased if acetate or propionate levels rose, indicating metabolism shifted towards hydrogen consumption by homoacetogenesis or propionate producers. These shifts occurred both at pH 4.5 and 5.2. Sparging the reactor with nitrogen to reduce hydrogen in the off-gas from 50% to 7% gave stable operation with a hydrogen yield of 1.9 moles hydrogen /mole hexose consumed over an 18-day period.     

Characterization of a hydrogen-producing granular sludge

This study demonstrated that hydrogen-producing acidogenic sludge could agglutinate into granules in a well-mixed reactor treating a synthetic sucrose-containing wastewater at 26 degrees C, pH 5.5, with 6 h of hydraulic retention. A typical matured granule is 1.6 mm in diameter, 1.038 g/mL in density, 11% in ash content, and over 50 m/h in settling velocity. Treating a solution containing 12.15 g/L of sucrose at a volumetric loading rate of 48.6 g/(L x d), the reactor containing 20 g/L of granular sludge degraded 97% of sucrose. Effluent comprised 46% acetate and 49% butyrate and the methane-free biogas comprised 63% hydrogen, 35% carbon dioxide, and 2% nitrogen. Hydrogen production rate was 13.0 L/(L x d), and the yield was 0.28 L/g-sucrose. The granule had multiple cracks on the surface and comprised two morphological types of bacteria: fusiform bacilli and a spore-forming bacterium. Phylogenetic analysis showed that 69.1% of the clones were affiliated with four Clostridium species in the family Clostridiaceae, and 13.5% with Sporolactobacillus racemicus in the Bacillus/Staphylococcus group.     

Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor

A novel bioreactor containing self-flocculated anaerobic granular sludge was developed for high-performance hydrogen production from sucrose-based synthetic wastewater. The reactor achieved an optimal volumetric hydrogen production rate of approximately 7.3 L/h/L (7,150 mmol/d/L) and a maximal hydrogen yield of 3.03 mol H2/mol sucrose when it was operated at a hydraulic retention time (HRT) of 0.5 h with an influent sucrose concentration of 20 g COD/L. The gas-phase hydrogen content and substrate conversion also exceeded 40 and 90%, respectively, under optimal conditions. Packing of a small quantity of carrier matrices on the bottom of the upflow reactor significantly stimulated sludge granulation that can be accomplished within 100 h. Among the four carriers examined, spherical activated carbon was the most effective inducer for granular sludge formation. The carrier-induced granular sludge bed (CIGSB) bioreactor was started up with a low HRT of 4-8 h (corresponding to an organic loading rate of 2.5-5 g COD/h/L) and enabled stable operations at an extremely low HRT (up to 0.5 h) without washout of biomass. The granular sludge was rapidly formed in CIGSB supported with activated carbon and reached a maximal concentration of 26 g/L at HRT = 0.5 h. The ability to maintain high biomass concentration at low HRT (i.e., high organic loading rate) highlights the key factor for the remarkable hydrogen production efficiency of the CIGSB processes.     

Start‐up of Anaerobic Hydrogen Producing Reactors Seeded with Sewage Sludge

The procedure for starting‐up continuously stirred tank reactors (CSTR) for acclimating anaerobic hydrogen‐producing microorganisms with sewage sludge was investigated. Initially, feeding with glucose and sucrose as well as mixing were carried out in semicontinuous mode; hydraulic retention time (HRT) was in an order of 20, 15, 10, 5, 2.5 and 2 days. When the pH declined to its lowest value (pH 5.18), it was adjusted to 6.7 using sodium hydroxide (1 N). At the same time, the semi‐continuous operation was changed to a continuous one. Finally, the pH was continuously regulated at approximately 6.7. The results indicate that this procedure can be used to cultivate seed sludge for hydrogen production from sewage sludge resulting in a large hydrogen production in less than 60 days. When the substrate was glucose, a hydrogen yield of 1.63 mol H₂/mol glucose and a specific hydrogen production rate of 321 mmol H₂/g VSS day at an HRT of 13.3 h was achieved. When the substrate was sucrose with the same HTR, a hydrogen yield of 4.45 mol H2/mol sucrose and a specific hydrogen production rate of 707 mmol H₂/g VSS day was obtained.     

Biohydrogen production from glucose in upflow biofilm reactors with plastic carriers under extreme thermophilic conditions (70 degrees C)

Biohydrogen could efficiently be produced in glucose-fed biofilm reactors filled with plastic carriers and operated at 70 degrees C. Batch experiments were, in addition, conducted to enrich and cultivate glucose-fed extreme-thermophilic hydrogen producing microorganisms from a biohydrogen CSTR reactor fed with household solid waste. Kinetic analysis of the biohydrogen enrichment cultures show that substrate (glucose) likely inhibited hydrogen production when its concentration was higher than 1 g/L. Different start up strategies were applied for biohydrogen production in biofilm reactors operated at 70 degrees C, and fed with synthetic medium with glucose as the only carbon and energy source. A biofilm reactor, started up with plastic carriers, that were previously inoculated with the enrichment cultures, resulted in higher hydrogen yield (2.21 mol H(2)/mol glucose consumed) but required longer start up time (1 month), while a biofilm reactor directly inoculated with the enrichment cultures reached stable state much faster (8 days) but with very low hydrogen yield (0.69 mol H(2)/mol glucose consumed). These results indicate that hydraulic pressure is necessary for successful immobilization of bacteria on carriers, while there is the risk of washing out specific high yielding bacteria.     

Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge

A novel continuously stirred anaerobic bioreactor (CSABR) seeded with silicone-immobilized sludge was developed for high-rate fermentative H2 production using sucrose as the limiting substrate. The CSABR system was operated at a hydraulic retention time (HRT) of 0.5-6 h and an influent sucrose concentration of 10-40 g COD/L. With a high feeding sucrose concentration (i.e., 30-40 g COD/L) and a short HRT (0.5 h), the CSABR reactor produced H2 more efficiently with the highest volumetric rate (VH2) of 15 L/h/L (i.e., 14.7 mol/d/L) and an optimal yield of ca. 3.5 mol H2/mol sucrose. The maximum VH2 value obtained from this work is much higher than any other VH2 values ever documented. Formation of self-flocculated granular sludge occurred during operation at a short HRT. The granule formation is thought to play a pivotal role in the dramatic enhancement of H2 production rate, because it led to more efficient biomass retention. A high biomass concentration of up to 35.4 g VSS/L was achieved even though the reactor was operated at an extremely low HRT (i.e., 0.5 h). In addition to gaining high biomass concentrations, formation of granular sludge also triggered a transition in bacterial community structure, resulting in a nearly twofold increase in the specific H2 production rate. According to denatured-gradient-gel-electrophoresis analysis, operations at a progressively decreasing HRT resulted in a decrease in bacterial population diversity. The culture with the best H2 production performance (at HRT = 0.5 h and sucrose concentration = 30 g COD/L) was eventually dominated by a presumably excellent H2-producing bacterial species identified as Clostridium pasteurianum.     

High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring

Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.     

Effects of initial lactic acid concentration, HRTs, and OLRs on bio-hydrogen production from lactate-type fermentation

A batch test and continuous operation were performed to identify the effect of lactate on hydrogen production at pH 4.5. When the initial lactic acid concentration was increased from 0 to 8 g/L in the batch test, the hydrogen yield also increased from 1.41 to 1.72 mol-H₂/mol-glucose. The system exhibited a long lag time and an insignificant hydrogen yield with 16 g-lactic acid/L. A continuous stirred tank reactor (CSTR) was operated at different organic loading rates (OLRs: 10, 15, 20 and 40 g/L/day) and hydraulic retention times (HRTs: 6, 12 and 24 h). At an OLR of 20 g-glucose/L/day and 12 h of HRT, the hydrogen yield was 1.2 mol-H₂/mol-glucose. The yield decreased with a 24 h HRT. Even though lactate was one of the major constituents of volatile fatty acids (VFAs), hydrogen production was feasible throughout the operation. Clostridium sp. was the dominant hydrogen-producing bacteria in the system.     

Anaerobic Hydrogen Production from Sucrose Using an Acid‐Enriched Sewage Sludge Microflora

A novel and high‐rate anaerobic sequencing bath reactor (ASBR) process was used to evaluate the hydrogen productivity of an acid‐enriched sewage sludge microflora at a temperature of 35 °C. In this ASBR process a 4 h cycle, including feed, reaction, settle, and decant steps, was repeatedly performed in a 5 L reactor. The sucrose substrate concentration was 20 g COD/L; the hydraulic retention time (HRT) was maintained at 12–120 h at the initial period and thereafter at 4–12 h. The reaction/settle period ratio, which is the most important parameter for ASBR operation was 1.7. The experimental results indicated that the hydrogenic activity of the sludge microflora was HRT‐dependent and that proper pH control was necessary for a stable operation of the bioreactor. The peak hydrogenic activity value was attained at an HRT of 8 h and an organic loading rate of 80 kg COD/m³ × day. Each mole of sucrose in the reactor produced 2.8 mol of hydrogen and each gram of biomass produced 39 mmol of hydrogen per day. An overly‐short HRT might deteriorate the hydrogen productivity. The concentration ratios of butyric acid to’acetic acid, as well as volatile fatty acid and soluble microbial products to alkalinity can be used as monitoring indicators for the hydrogenic bioreactor.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Fedbatch operation using Clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production

We demonstrated the feasibility of fedbatch operation using Clostridium acetobutylicum suspension culture as a biocatalyst for the continuous production of hydrogen. The optimum operating pH and temperature of the current cultivation system for hydrogen production were pH 6.0 and 37 degrees C, respectively. The volumetric loading of the bioreactor for hydrogen production can be as high as 650 mmol hydrogen/L culture with a yield at approximately 2.0 mol hydrogen/mol glucose. Acetate and butyrate made up approximately 80% of the total metabolites. The inhibitory effect from the two metabolites on the hydrogen production process was investigated. Butyrate at a concentration higher than 13 g/L significantly inhibited not only cell growth but also hydrogen production (i.e., specific hydrogen production rate). Acetate appears to be less toxic than butyrate to the hydrogen production process. While significantly inhibiting cell growth, acetate hardly affected hydrogen production. Finally, the factors limiting cultivation performance were discussed and possible strategies for enhancing the production of hydrogen were proposed.     

Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture

Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70°C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 ± 5.2 mL‐H₂/g‐sugars (14.2 ± 0.2 mmol‐H₂/g‐sugars) at the hydrolysate concentration of 5% (v/v). Continuously fed, and the continuously stirred tank reactor (CSTR), operating at 3 day hydraulic retention time (HRT) and fed with 20% (v/v) hydrolysate could successfully produce hydrogen. The hydrogen yield and production rate were 178.0 ± 10.1 mL‐H₂/g‐sugars (7.9 ± 0.4 mmol H₂/g‐sugars) and 184.0 ± 10.7 mL‐H₂/day L_reactor (8.2 ± 0.5 mmol‐H₂/day L_reactor), respectively, corresponding to 12% of the chemical oxygen demand (COD) from sugars. Additionally, it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were effectively degraded in the CSTR, and their concentrations were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were phylogenetically related to the Caldanaerobacter subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum. Biotechnol. Bioeng. 2010;105: 899–908. © 2009 Wiley Periodicals, Inc.     

Performance and population analysis of a non‐sterile trickle bed reactor inoculated with Caldicellulosiruptor saccharolyticus,a thermophilic hydrogen producer

Non‐axenic operation of a 400 L trickle bed reactor inoculated with the thermophile Caldicellulosiruptor saccharolyticus, yielded 2.8 mol H₂/mol hexose converted. The reactor was fed with a complex medium with sucrose as the main substrate, continuously flushed with nitrogen gas, and operated at 73°C. The volumetric productivity was 22 mmol H₂/(L filterbed h). Acetic acid and lactic acid were the main by‐products in the liquid phase. Production of lactic acid occurred when hydrogen partial pressure was elevated above 2% and during suboptimal fermentation conditions that also resulted in the presence of mono‐ and disaccharides in the effluent. Methane production was negligible. The microbial community was analyzed at two different time points during operation. Initially, other species related to members of the genera Thermoanaerobacterium and Caldicellulosiruptor were present in the reactor. However, these were out‐competed by C. saccharolyticus during a period when sucrose was completely used and no saccharides were discharged with the effluent. In general, the use of pure cultures in non‐sterile industrial applications is known to be less useful because of contamination. However, our results show that the applied fermentation conditions resulted in a culture of a single dominant organism with excellent hydrogen production characteristics. Biotechnol. Bioeng. 2009;102: 1361–1367. © 2008 Wiley Periodicals, Inc.     

Continuous fermentation of feed streams containing D-glucose and D-xylose in a two-stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,(1,2,3) To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y-2460 and Saccharomyces cerevisiae NRRL Y-2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y-2235 fermented glucose but not xylose. Y-2460 consumed both sugars but fermented glucose inefficiently relative to Y-2235, and it suffered a diauxic lag lasting 10-20 h when given a sugar mixture. Immobilized Y-2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y-2460 exhibited an optimum productivity at 30-40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y-2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y-2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8-g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y-2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y-2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two-stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y-2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y-2235 plug flow reactor would enrich the hemicellulose-derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.     

Anaerobic co-digestion of desugared molasses with cow manure; focusing on sodium and potassium inhibition

Desugared molasses (DM), a syrup residue from beet-molasses, was investigated for biogas production in both batch and in continuously-stirred tank reactor (CSTR) experiments. DM contained 2-3 times higher concentration of ions than normal molasses, which could inhibit the biogas process. The effect of sodium and potassium concentration on biogas production from manure was also investigated. Fifty percent inhibition occurred at sodium and potassium concentration of 11 and 28 g/L, respectively. The reactor experiments were carried out to investigate the biogas production from DM under different dilutions with water and co-digestion with manure. Stable operation at maximum methane yield of 300 mL-CH₄/gVS-added was obtained at a mixture of 5% DM in cow manure. The biogas process was inhibited at DM concentrations higher than 15%. Manure was a good base substrate for co-digestion, and a stable anaerobic digestion could be achieved by co-digesting DM with manure at the concentration below 15% DM.     

Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.     

Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase

Gluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose-fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross-linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin-immobilized inulinase in alginate beads. The optimum amounts of both chitin-immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h(-1) and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed-bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h(-1) and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed-bed reactor without any loss of activity, while half-life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.     

Hydrolysis of liquefied corn starch in a membrane reactor

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first-order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (V(m)) was 3.86 g glucose/L min and the apparent Michaelis constant (K(m) (')) was 562 g/L. The K(m) (') value for the continuous reactor was 2-7 times greater than that obtained in a batch reactor.Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.     

Thermophilic biohydrogen production from glucose with trickling biofilter

Thermophilic H2 production from glucose was studied at 55-64 degrees C for 234 days using a continuous trickling biofilter reactor (TBR) packed with a fibrous support matrix. Important parameters investigated included pH, temperature, hydraulic retention time (HRT), and glucose concentration in the feed. The optimal pH and temperature were 5.5 and 60 degrees C, respectively. With decreasing HRT or increasing inlet glucose concentration, volumetric H2 production rate increased but the H2 production yield to glucose decreased gradually. The biogas composition was almost constant at 53 +/- 4% (v/v) of H2 and 47 +/- 4% (v/v) of CO2. No appreciable CH4 was detected when the reactor was under a normal operation. The carbon mass balance showed that, in addition to cell mass, lactate, n-butyrate, CO2, and acetate were major products that comprised more than 85% of the carbon consumed. The maximal volumetric H2 production rate and H2 yield to glucose were 1,050 +/- 63 mmol H2/l.d and 1.11 +/- 0.12 mol H2/mol glucose, respectively. These results indicate that the thermophilic TBR is superior to most suspended or immobilized reactor systems reported thus far. This is the first report on continuous H2 production by a thermophilic TBR system.