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.     

Thermophilic production of lactic acid using integrated membrane bioreactor systems coupled with monopolar electrodialysis

The thermophilic Bacillus strain BS119 was selected for this study to demonstrate the long term performance of lactic acid production and simultaneous pre-purification. Integrated continuous cell recycle cultivation using ultra-filtration membrane bioreactor (MBR) systems was investigated. The permeate from the MBR was routed to an on-line electrodialysis (ED) to recover, pre-purify and concentrate lactate. The cultivation and ED was operated at 60 degrees C for more than 1,000 h at a pH of 6.5. At lower dilution rate (0.02 h(-1)), lactate concentration reached a maximum of 55 g l(-1) with clearly lower residual glucose levels. At 0.04 h(-1), lactate concentration was significantly lower at 35 g l(-1). Maximal volumetric productivities of 1.38 g l(-1) h(-1) were achieved. Under stable conditions, lactic acid yield on consumed glucose appeared stable at around 80%. It could be demonstrated that the addition of supplements like yeast extract and peptone severely influences product formation. Integration of mono-polar ED with the MBR systems yields lactate solutions with concentrations of up to 115 g l(-1). Because of the low substrate feed concentrations (less than 50 g l(-1)), lactate flux was rather poor, reaching a low maximum of 140 g m(-2) h(-1); nevertheless, stack energy consumption was positive with an average of 0.49 kWh kg(-1) lactate.     

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.     

Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process

A fermentative hydrogen-producing bacterium, Clostridium tyrobutyricum JM1, was isolated from a food waste treating process using 16S rRNA gene sequencing and amplified ribosomal DNA restriction analysis (ARDRA). A fixed-bed bioreactor packed with polyurethane foam as support matrix for the growth of the isolate was operated at different hydraulic retention time (HRT) to evaluate its performance for hydrogen production. The reactor achieved the maximal hydrogen production rate of 7.2 l H(2)l(-1)d(-1) at 2h HRT, where hydrogen content in biogas was 50.0%, and substrate conversion efficiency was 97.4%. The maximum hydrogen yield was 223 ml (g-hexose)(-1) with an influent glucose concentration of 5 g l(-1). Therefore, the immobilized reactor using C. tyrobutyricum JM1 was an effective and stable system for continuous hydrogen production.     

Simultaneous nitrification-denitrification achieved by an innovative internal-loop airlift MBR: comparative study

This study assessed the performance of different single-stage continuous aerated submerged membrane bioreactors (MBR) for nitrogen removal. Almost complete nitrification was achieved in each MBR irrespective of operating mode and biomass system. Denitrification was found to be the rate-limiting step for total nitrogen (T-N) removal. The MBR with internal-loop airlift reactor (ALR) configuration performed better as regards T-N removal compared with continuous stirred-tank reactor (CSTR). It was demonstrated that simultaneous nitrification and denitrification (SND) is the mechanism leading to nitrogen removal and the contribution of microenvironment on SND is more remarkable for the MBRs with hybrid biomass. Macroenvironment analyses showed that gradient distribution of dissolved oxygen (DO) level in airlift MBRs imposed a significant effect on SND. Higher mixed liquor suspended solid (MLSS) concentration led to the improvement in T-N removal by enhancing anoxic microenvironment. Apparent nitrite accumulation coupled with higher nitrogen reduction was accomplished at MLSS concentration exceeded 12.6 g/L.     

Performance of an anaerobic bioreactor with biomass recycling, continuously removing COD and sulphate from industrial wastes

A 30-l anaerobic bioreactor with biomass recycling was used to provide a continuous reduction in sulphate and a continuous COD removal from wastewater, which consisted of the effluent from an industrial pig fattening farm, enriched with technical FeSO(4) x 7H(2)O, a waste product from ferrous metallurgy. The concentrations of sulphate and COD in the wastewater amounted to 2.73 g l(-1) and 3.15 g l(-1), respectively. The HRT (hydraulic retention time) of 10-1.7d produced an extent of sulphate and COD reduction which totalled 98% and 88%, respectively. When the HRT was further shortened, the efficiency of reduction in sulphate and COD decreased. The maximum removal rate constants for both the pollutants, calculated by means of a modified Stover-Kincannon model, were 80.9 g COD l(-1)d(-1) and 41.8 g SO(4)(2-)l(-1)d(-1), the values of the saturation constants being 91.582 g COD l(-1)d(-1) and 42.398 g SO(4)(2-)l(-1)d(-1).     

Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. III. Utilization of two thermostable beta-glycosidases in a continuous ultrafiltration membrane reactor and galacto-oligosaccharide formation under steady-state conditions.

Hydrolysis of lactose by hyperthermophilic beta-glycosidases from the archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) was carried out at 70 degrees C in a continuous stirred-tank reactor (CSTR) coupled to a 10-kDa cross-flow ultrafiltration module to recycle the enzyme. Recirculation rates of > or =1 min(-1), reaction of proteins with reducing sugars, and enzyme adsorption onto the membrane are major "operational" factors of enzyme inactivation in the CSTR. They cause the half-life times of SsbetaGly and CelB to be reduced two- and eight-fold, respectively, the average value for both enzymes now being approximately 5 to 7 days. Using lactose at initial concentrations of 45 and 170 g/L, the CSTR was operated at a constant conversion level of approximately 80% for more than 2 weeks without the occurrence of microbial contamination. The productivities for the SsbetaGly-catalyzed conversion of lactose were determined at different dilution rates and initial substrate concentrations, and exceed by a factor of < or =2 those observed with CelB under otherwise identical conditions. This difference reflects the approximately eight-fold stronger product inhibition of CelB by D-glucose. While the maximum total galacto-oligosaccharide production (90-100 mM) at 170 g/L lactose in the CSTR was not different from that in the batch reactor (CelB) or was greater by approximately 25% (SsbetaGly), continuous and batchwise reactions with both enzymes differed markedly with regard to relative proportions of the individual saccharide components present at 80% substrate conversion. The CSTR yielded an up to four-fold greater ratio of disaccharides to trisaccharides concomitant with a 5- to 30-fold larger relative proportion of beta-D-Galp-(1-->3)-D-Glc in the product mixture. The results show that apart from continuous hydrolysis of lactose at 70 degrees C, a CSTR charged with SsbetaGly or CelB and operated at steady-state conditions could be a useful reaction system for the production of galacto-oligosaccharides in which composition is narrower and more easily programmable, in terms of the individual components contained, as compared to the batchwise reaction.     

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.     

The effect of organic loading rate and retention time on hydrogen production from a methanogenic CSTR

The possibility of shifting a methanogenic process for hydrogen production by changing the process parameters viz., organic loading rate (OLR) and hydraulic retention time (HRT) was evaluated. At first, two parallel semi-continuously fed continuously stirred tank reactors (CSTR) were operated as methanogenic reactors (M1 and M2) for 78 days. Results showed that a methane yield of 198-218 L/kg volatile solids fed (VS(fed)) was obtained when fed with grass silage at an OLR of 2 kgVS/m3/d and HRT of 30 days. After 78 days of operation, hydrogen production was induced in M2 by increasing the OLR from 2 to 10 kgVS/m3/d and shortening the HRT from 30 to 6 days. The highest H? yield of 42 L/kgVS(fed) was obtained with a maximum H? content of 24%. The present results thus demonstrate that methanogenic process can be shifted towards hydrogen production by increasing the OLR and decreasing HRT.     

Continuous production of L-tryptophan from indole and L-serine by immobilized Escherichia coli cells

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Use of fixed film and CSTR reactor for anaerobic treatment of stillage of wood hydrolysate

Summary The application of fixed film and CSTR reactor-systems for the treatment of stillage from wood hydrolysates was studied in laboratory units at 35° and 55°C. Organic loads of 10.7 (10) gCOD/l/d with COD-reduction of 86.6 (84.4)% and a biogas production rate of 400 (380) ml/ gCOD with 63 (62) % methane were feasible using fixed film systems, whereas organic loads with CSTR reactor-systems were only 2.4 gCOD/l/day.     

Advanced anaerobic bioconversion of lignocellulosic waste for bioregenerative life support following thermal water treatment and biodegradation by Fibrobacter succinogenes

The feasibility of nearly-complete conversion of lignocellulosic waste (70% food crops, 20% faecal matter and 10% green algae) into biogas was investigated in the context of a life support project. The treatment comprised a series of processes, i.e., a mesophilic laboratory scale CSTR (continuously stirred tank reactor), an upflow biofilm reactor, a fiber liquefaction reactor employing the rumen bacterium Fibrobacter succinogenes and a hydrothermolysis system in near-critical water. By the one-stage CSTR, a biogas yield of 75% with a specific biogas production of 0.37 l biogas g(-1) VSS (volatile suspended solids) added at a RT (hydraulic retention time) of 20-25 d was obtained. Biogas yields could not be increased considerably at higher RT, indicating the depletion of readily available substrate after 25 d. The solids present in the CSTR-effluent were subsequently treated in two ways. Hydrothermal treatment (T approximately 310-350 degrees C, p approximately 240 bar) resulted in effective carbon liquefaction (50-60% without and 83% with carbon dioxide saturation) and complete sanitation of the residue. Application of the cellulolytic Fibrobacter succinogenes converted remaining cellulose contained in the CSTR-effluent into acetate and propionate mainly. Subsequent anaerobic digestion of the hydrothermolysis and the Fibrobacter hydrolysates allowed conversion of 48-60% and 30%, respectively. Thus, the total process yielded biogas corresponding with conversions up to 90% of the original organic matter. It appears that particularly mesophilic digestion in conjunction with hydrothermolysis at near-critical conditions offers interesting features for (nearly) complete and hygienic carbon and energy recovery from human waste in a bioregenerative life support context.     

Optimisation of biogas production from manure through serial digestion: lab-scale and pilot-scale studies

In the present study, the possibility of optimizing biogas production from manure by serial digestion was investigated. In the lab-scale experiments, process performance and biogas production of serial digestion, two methanogenic continuously stirred tank reactors (CSTR) connected in series, was compared to a conventional one-step CSTR process. The one-step process was operated at 55 degrees C with 15d HRT and 5l working volume (control). For serial digestion, the total working volume of 5l was distributed as 70/30%, 50/50%, 30/70% or 13/87% between the two methanogenic reactors, respectively. Results showed that serial digestion improved biogas production from manure compared to one-step process. Among the tested reactor configurations, best results were obtained when serial reactors were operated with 70/30% and 50/50% volume distribution. Serial digestion at 70/30% and 50/50% volume distribution produced 13-17.8% more biogas and methane and, contained low VFA and residual methane potential loss in the effluent compared to the one-step CSTR process. At 30/70% volume distribution, an increase in biogas production was also noticed but the process was very unstable with low methane production. At 13/87% volume distribution, no difference in biogas production was noticed and methane production was much lower than the one-step CSTR process. Pilot-scale experiments also showed that serial digestion with 77/23% volume distribution could improve biogas yields by 1.9-6.1% compared to one-step process. The study thus suggests that the biogas production from manure can be optimized through serial digestion with an optimal volume distribution of 70/30% or 50/50% as the operational fluctuations are typically high during full scale application. However, process temperature between the two methanogenic reactors should be as close as possible in order to derive the benefits of serial coupling.     

Effect of hydraulic retention time on anaerobic hydrogenesis in CSTR

The objective of this work was to evaluate the production of hydrogen in a continuous system as a function of hydraulic retention time (HRT). The intermediates accumulated and other parameters of pH, oxidation-reduction potential were quantified. The heat treatment (103 degrees C for 24 h) of the compost from a cattle dung composting facility was able to select H2-producing spores; this product was used as a seed for continuous systems. The brewery waste was used as substrate. For the eight runs with combinations of five HRTs and four pHs, the results indicate that at pH=5.5, a maximum H2 production of 47% H2 concentration, 43 ml H2/g COD(added), and 3.1 l H2/l reactor d was achieved at HRT=18 h. Nevertheless, at HRT=18 h, pH 5.5 was also the optimum pH for the maximum H2 production among four pHs evaluated from 5 to 6.5. There was a significant accumulation of volatile acid and alcohols during the entire study.     

Metabolic pathways of hydrogen production in fermentative acidogenic microflora

Biohydrogen production from organic wastewater by anaerobically activated sludge fermentation has already been extensively investigated, and it is known that hydrogen can be produced by glucose fermentation through three metabolic pathways, including the oxidative decarboxylation of pyruvic acid to acetyl-CoA, oxidation of NADH to NAD+, and acetogenesis by hydrogen-producing acetogens. However, the exact or dominant pathways of hydrogen production in the anaerobically activated sludge fermentation process have not yet been identified. Thus, a continuous stirred-tank reactor (CSTR) was introduced and a specifically acclimated acidogenic fermentative microflora obtained under certain operation conditions. The hydrogen production activity and potential hydrogen-producing pathways in the acidogenic fermentative microflora were then investigated using batch cultures in Erlenmeyer flasks with a working volume of 500 ml. Based on an initial glucose concentration of 10 g/l, pH 6.0, and a biomass of 1.01 g/l of a mixed liquid volatile suspended solid (MLVSS), 247.7 ml of hydrogen was obtained after a 68 h cultivation period at 35 +/- 1 degrees C. Further tests indicated that 69% of the hydrogen was produced from the oxidative decarboxylation of pyruvic acid, whereas the remaining 31% was from the oxidation of NADH to NAD+. There were no hydrogen-producing acetogens or they were unable to work effectively in the anaerobically activated sludge with a hydraulic retention time (HRT) of less than 8 h.     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.     

Xylitol production by <Emphasis Type="Italic">Candida tropicalis</Emphasis> in a chemically defined medium

A chemically defined medium that included urea (5 g l(-1)) as a nitrogen source and various vitamins was substituted for a complex medium containing yeast extract (10 g l(-1)) in the production of xylitol by Candida tropicalis. In a fed-batch culture with the chemically defined medium, 237 g xylitol l(-1) was produced from 270 g xylose l(-1) after 120 h. The volumetric rate of xylitol production and the xylitol yield from xylose were 2 g l(-1) h(-1) and 89%, respectively. These values were about 5% lower and 4% higher, respectively, than those obtained using the complex medium. These results indicate that xylitol can be produced effectively in a chemically defined medium.     

Reduction in excess sludge production in a dairy wastewater treatment plant via nozzle-cavitation treatment: Case study of an on-farm wastewater treatment plant

Nozzle-cavitation treatment was used to reduce excess sludge production in a dairy wastewater treatment plant. During the 450-d pilot-scale membrane bioreactor (MBR) operation, when 300 l of the sludge mixed liquor (1/10 of the MBR volume) was disintegrated per day by the nozzle-cavitation treatment with the addition of sodium hydrate (final concentration: 0.01% W/W) and returned to the MBR, the amount of excess sludge produced was reduced by 80% compared with that when sludge was not disintegrated.