Continuous production of α-amylase byBacillus amyloliquefaciens in a two-stage fermentor

Summary The production of a secondary metabolite (-amylase) by a highly aerobic bacterium (Bacillus amyloliquefaciens) was examined in batch, single-stage chemostat, two-stage stirred tank, and two-stage stirred tank/tubular reactor configurations. The relative performance of these reactor systems as measured by product concentration and volumetric productivity was compared, and the effect of aeration rate on the extent of plug flow in the tubular reactor was examined.     

Rhodobacter sphaeroides RV cultivation and hydrogen production in a one- and two-stage chemostat

Rhodobacter sphaeroides RV cultivation and hydrogen production were studied in a one- and two-stage chemostat using lactic acid as substrate. Light saturation was observed when light intensities equal to or above 10 klx were applied. Under light saturation, the two-stage chemostat appeared to be very effective for hydrogen production, allowing complete nitrogen removal by bacterial growth in the first reactor. The hydrogen evolution rate in the second reactor was up to 75 ml H₂ (g dry weight)^–1 h^–1. Accumulation of storage material was observed in the second reactor of the two-stage chemostat under a large carbon excess and limiting light irradiance. The optimal hydraulic residence time was 15 h for both stages, leading to a total hydrogen production about 40% higher than in the one-stage chemostat. Under increasing influent ammonium and yeast extract concentrations, opposite trends of decreasing bacterial activity and increasing concentration resulted in a linear increase of the overall hydrogen production to 1.4–1.6lH₂ (l reactor)^–1 day^–1. Hydrogen production quickly fell when nitrogen was not completely metabolised. The hydrogen evolution rate was also found to depend on lactic acid concentration, and maximum bacterial activity was observed at 100 mM influent lactic acid.     

Reducing the risk of foaming and decreasing viscosity by two-stage anaerobic digestion of sugar beet pressed pulp

Anaerobic digestion (AD) of sugar beet pressed pulp (SBPP) is a promising treatment concept. It produces biogas as a renewable energy source making sugar production more energy efficient and it turns SBPP from a residue into a valuable resource. In this study one- and two-stage mono fermentation at mesophilic conditions in a continuous stirred tank reactor were compared. Also the optimal incubation temperature for the pre-acidification stage was studied. The fastest pre-acidification, with a hydraulic retention time (HRT) of 4 days, occurred at a temperature of 55 °C. In the methanogenic reactor of the two-stage system stable fermentation at loading rate of 7 kg VS/m³ d was demonstrated. No artificial pH adjustment was necessary to maintain optimum levels in both the pre-acidification and the methanogenic reactor. The total HRT of the two-stage AD was 36 days which is considerably lower compared to the one-stage AD (50 days). The frequently observed problem of foaming at high loading rates was less severe in the two-stage reactor. Moreover the viscosity of digestate in the methanogenic stage of the two-stage fermentation was in average tenfold lower than in the one-stage fermentation. This decreases the energy input for the reactor stirring about 80 %. The observed advantages make the two-stage process economically attractive, despite higher investments for a two reactor system.     

The effect of maltose on dextran yield and molecular weight distribution

Dextran synthesis has been studied since the Second World War, when it was used as blood plasma expander. This polysaccharide composed of glucose units is linked by an α-1,6-glucosidic bond. Dextransucrase is a bacterial extra cellular enzyme, which promotes the dextran synthesis from sucrose. When, besides sucrose, another substrate (acceptor) is also present in the reactor, oligosaccharides are produced and part of the glucosyl moieties from glucose is consumed to form these acceptor products, decreasing the dextran yield. Although dextran enzymatic synthesis has been extensively studied, there are few published studies regarding its molecular weight distribution. In this work, the effect of maltose on yield and dextran molecular weight synthesized using dextransucrase from Leuconostoc mesenteroides B512F, was investigated. According to the obtained results, maltose is not able to control and reduce dextran molecular weight distribution and synthesis carried out with or without maltose presented the same molecular weight distribution profile.     

Aerobic treatment of dairy wastewater with sequencing batch reactor systems

Performances of single-stage and two-stage sequencing batch reactor (SBR) systems were investigated for treating dairy wastewater. A single-stage SBR system was tested with 10,000 mg/l chemical oxygen demand (COD) influent at three hydraulic retention times (HRTs) of 1, 2, and 3 days and 20,000 mg/l COD influent at four HRTs of 1, 2, 3, and 4 days. A 1-day HRT was found sufficient for treating 10,000-mg/l COD wastewater, with the removal efficiency of 80.2% COD, 63.4% total solids, 66.2% volatile solids, 75% total Kjeldahl nitrogen, and 38.3% total nitrogen from the liquid effluent. Two-day HRT was believed sufficient for treating 20,000-mg/l COD dairy wastewater if complete ammonia oxidation is not desired. However, 4-day HRT needs to be used for achieving complete ammonia oxidation. A two-stage system consisting of an SBR and a complete-mix biofilm reactor was capable of achieving complete ammonia oxidation and comparable carbon, solids, and nitrogen removal while using at least 1/3 less HRT as compared to the single SBR system.     

Continuous production of acrylamide using immobilizedBrevibacterium sp. CH2 in a two-stage packed bed reactor

Summary Acrylamide was enzymatically produced from acrylonitrile (AN) in a two-stage packed bed reactor with immobilizedBrevibacterium sp. CH2 cells. Continuous production became possible because of higher tolerance of CH2 nitrile hydratase with respect to 6% AN than either the CH1 enzyme or theCorynebacterium enzyme of Nitto Chemical Industry which rapidly deactivated in a solution of 3% AN. The maximum volumetric productivity of 93.2 g/(L.h) was obtained when the reactor was fed with 6% AN at 4 °C, which compares that of 8.3 g/(L.h) with CH1 cells.     

Biofilm formation by exopolysaccharide mutants of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Mixed culture fermentation for the production of clinical quality dextran with starch and sucrose

Summary Mixed culture fermentations containing a dextranase-producing yeast, Lipomyces starkeyi, and a dextransucrase-producing bacterium, Leuconostoc mesenteroides, produced dextrans of selected size using sucrose and starch. Dextran and starch hydrolyzates, produced by a Lipomyces starkeyi dextranase and amylase, were incorporated into the dextran and formed small size dextran (Mr = 40kDa). The yields of total and clinical dextran in mixed culture fermentation using 12% sucrose and 3% starch were higher, 84 and 79% of theoretical yield, than those using 15% sucrose only, 73 and 69% of theoretical yield, respectively.     

Production of rosmarinic acid from perfusion culture ofAnchusa officinalis in a membrane-aerated bioreactor

Summary In this study, a perfusion fermentation ofAnchusa officinalis was carried out in a stirred tank bioreactor integrated with an internal cross-flow filter. Bubble-free aeration via microporous membrane fibers was used to provide oxygen. A two-stage culture was successfully conducted in this reactor without filter fouling. In a 17 day fermentation, a cell density of 26 g dw/I and a rosmarinic acid productivity of 94 mg/l day were achieved. This productivity is three times that obtained in a batch culture.     

Two-stage biogas production by co-digesting molasses wastewater and sewage sludge

We evaluated the feasibility of co-digesting molasses wastewater and sewage sludge in a two-stage hydrogen- and methane-producing system. The highest energy was recovered at the 21-h hydraulic retention time (HRT) of the first hydrogenic reactor and at 56-h HRT of the secondary methanogenic reactor. Hence, the two-stage system recovered 1,822 kJ from 1 L of the mixed wastes (19.7: hydrogenic reactor plus, 1,802 kJ L^?1: methanogenic reactor). Despite the overloaded VFA-run with a short HRT of 56 h, the GAC-CH₄ reactor increased methane production rate and yields due to enhanced pH buffer capacity. An RNA-based community analysis showed that the Ethanoligenens and Methanosaeta dominated the hydrogen and methane bioreactor, respectively. The two-stage system of co-digesting molasses and sewage sludge is particularly cost-effective due to non-pretreatment of sewage sludge.     

A tubular segmented-flow bioreactor for the infection of insect cells with recombinant baculovirus

A continuous process of insect cell (S f9) growth and baculovirus infection is tested with the sequential combination of a CSTR and a tubular reactor. A tubular infection reactor enables continuous introduction of baculovirus and therefore avoids the ‘passage effect’ observed in two-stage CSTR systems. Moreover, a tubular reactor can be used to test cell infection kinetics and the subsequent metabolism of infected insect cells. Unlike batch and CSTR culture, cells in a horizontally positioned tubular reactor settle due to poor mixing. We have overcome this problem by alternately introducing air bubbles and media and by maintaining a linear velocity sufficient to keep cells suspended. This article addresses the development of the tubular reactor and demonstrates its use as an infection system that complements the two-stage CSTR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemical synthesis of polysaccharides. 7. Enzymatic hydrolysis of (1 → 6)-α-DL-glucopyranan (DL-dextran)

Hydrolysis of (1 → 6)-α-DL -glucopyranan (synthetic DL -dextran) by an endo-dextranase from a Penicillium species was examined in an acetate buffer solution (pH 5.3) at 37°C. Three samples of different tacticities (isotactic dyad content, 55, 63, and 72%) were employed with a clinical dextran for comparison. Colorimetric determination of the reducing end units of the saccharides produced during hydrolysis showed that the maximum degrees of hydrolysis based on the D-glucose units, (D.H.)_D, for the DL -dextrans were 21.4, 27.8, and 33.0% in the order of increasing isotacitic dyad content, whereas the (D.H.)_D value for the clinical dextran was 51.9%. A statistical treatment of the enzymatic hydrolysis is proposed to interpret the experimental results.     

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.     

Flavor Specificities of Satsuma Mandarin Juice Extracted by a New-type Screw Press Extraction System

Dextransucrases from Leuconostoc mesenteroides NRRL B-1416 and B-1375 strains were purified to electrophoretically homogeneous preparations. After successive column chromatographies, the enzyme fractions were treated with endodextranase, then subjected to preparative polyacrylamide gel electrophoresis. The purified dextransucrase from each strain had a dimeric structure of molecular weight 130,000~133,000. Alkaline treatment (pH 10.5) dissociated these dimer forms into the respective monomer forms having molecular weight of 64,000~68,000. The two enzymes were closely similar to each other in optimum conditions and thermal and pH stabilities. The purified B-1416 enzyme was activated 4.35-fold by the addition of exogenous dextran (0.5%), while the B-1375 enzyme was activated 2.76-fold. In the absence of exogenous dextran, both enzymes gave 5~10 min lag periods for reaction, which were abolished by the clinical dextran.     

Dextran anaphylactoid reaction in Sprague-Dawley CFY rats.

From a closed colony of randomly-bred Sprague-Dawley CFY rats about 23% failed to respond to intravenous dextran with the characteristic generalized anaphylactoid reaction, but still exhibited an inflammatory response when dextran was given into the foot pad. Brother-sister mating of rats showing the most expressed generalized reaction (reactor rats) yielded good responder offsprings, while the non-reactors had descendants completely unresponsive to systemic dextran. Brother-sister mating of selected non-reactor rats led to a gradual decrease in dextran paw oedema in the subsequent generations, and after the third mating, a complete local non-reactivity developed. In these rats the intradermal injection of dextran failed to increase vascular permeability, while the inflammatory response evoked by histamine, 5-HT, bradykinin, and compound 48/80 remained unchanged as compared to that of the reactor animals. These result show that the anaphylactoid reaction in Sprague-Dawley CFY rats is under genetic control.     

On the production of dextran by free and immobilized dextransucrase

Dextransucrase from Leuconostoc mesenteroides was produced in a semicontinuous culture with slow addition of a concentrated sucrose solution. The resulting high activity of the fermentation broth allowed a one-step purification method, by gel permeation chromatography (GPC) in 96.4% yield. This procedure resulted in 140-fold purification, with specific activity of 122 U/mg. The enzyme was immobilized onto an amino-Spherosil support activated with glutaraldehyde. Preparations with dextransucrase activities as high as 40.5 U/g of support were obtained, when low specific area supports were used and maltose was added during the enzyme coupling. Diffusional limitations were found during enzyme reaction, as shown by a kinetic study. As a consequence of immobilization, the average molecular weight of dextrans seems to increase. Immobilized dextransucrase looks promising for low-molecular-weight dextran production. Clinical dextran was synthesized when the polysaccharides produced in the presence of maltose were used as acceptor of a second synthesis reaction. The molecular weight distribution of the resulting production was less disperse than when clinical dextran was produced by acid hydrolysis of high-molecular-weight dextran.     

Fermentative production of dextran using Leuconostoc spp. isolated from fermented food products

Leuconostoc spp. （LSland LI1） isolated from sauerkraut and idli batter was selected for dextran production. To enhance the yield of dextran, effects of various parameters such as sucrose concentration, pH, temperature, incubation and inoculum percentage were analyzed. The optimum sucrose concentration for the Leuconostoc spp. （LS1 and LI1） was found to be 15% and 25% respectively. Isolates produced maximum dextran after 20 h of incubation at 29℃ and the optimum pH was found between 8 and 8.5. The inoculum concentration of 7.5% was more favorable for the production of dextran by Leuconostoc spp. （LS1 and LI1）. The growth kinetic parameters were studied and compared for the strains LS1 and LI1. Mass production of dextran was carried out using a stirred tank batch reactor. FTIR analysis was done to determine the functional groups of dextran, sephadex is prepared by cross linking dextran using epichlorohydrin and the functional groups are determined by FTIR analysis.     

Bioaugmentation of a two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion concept for improvement of the methane yield from cattle manure

The possibility of improving a two-stage (68 degrees C/55 degrees C) anaerobic digestion concept for treatment of cattle manure was studied. In batch experiments, a 10-24% increase of the specific methane yield from cattle manure and its fractions was obtained, when the substrates were inoculated with bacteria of the genus Caldicellusiruptor and Dictyoglomus. In a reactor experiment inoculation of a 68 degrees C pretreatment reactor with Caldicellusiruptor resulted in a 93% increase in the methane yield of the pretreatment reactor for a period of 18 days, but gave only a slight increase in the overall methane yield of the two-stage setup.     

Continuous production of scopolamine by a culture of Duboisia leichhardtii hairy root clone in a bioreactor system

The scopolamine-releasing hairy root clone DL47-1 of Duboisia leichhardtii was cultured in an Amberlite XAD-2 column-combined bioreactor system for continuous production of scopolamine. The medium used was continuously exchanged during culture to maintain the electrical conductivity of the medium constant. After culturing the hairy roots in the system for 11 weeks, 0.5 g/l of scopolamine was obtained in the column. When the roots were cultures in the reactor system containing polyurethane foam or stainless-steel mesh to support the hairy roots, scopolamine recovery was increased. Thereafter, a two-stage culture, the first stage in the medium for hairy root growth and the second stage in the medium for scopolamine release, was carried out in this system by using a turbine-blade reactor with stainless-steel mesh as a support. Under these conditions, 1.3 g/l of scopolamine was recovered during 11 weeks of culture in the medium for scopolamine release. This bioreactor system seems applicable for the production of various plant metabolites by cultures of hairy roots. Correspondence to: T. Muranaka     

Two-stage anaerobic treatment of dairy wastewater using HUASB with PUF and PVC carrier

In the present study, an attempt has been made to treat dairy wastewater entirely via anaerobic treatment over a period of 215 days, using two-stage Hybrid Upflow Anaerobic Sludge Blanket (HUASB) reactors, which offer the advantages associated both with fixed film and upflow sludge blanket treatments. A HUASB with polyurethane foam cubes was used for stage I, and a HUASB utilizing PVC-cut rings was used for stage II. The output from stage I was used as the input for stage II. The two-stage reactor was operated at an organic loading rate that varied from 10.7 to 21.4 kg COD m³/d for a period of 215 days, including the start-up period. The ideal organic loading rate for the two-stage reactor was 19.2 kg COD/m³/d. A further 21.4 kg COD m³/d increase in the organic loading rate resulted in the souring of the reactor function in stage I, which consequently reduced the overall reactor performance. Combined COD removal during the stable operation period (10.7 to 19.2 kg COD m³/d) occurred in a range between 97 and 99%. The methane content in the biogas varied from 65 to 70% in stage I, and from 63 to 66% in stage II. The two-stage anaerobic treatment using HUASB with PUF and PVC described in this work is expected to constitute a better alternative for the complete treatment of dairy wastewater than high-rate anaerobic, anaerobic/aerobic, and two-phase anaerobic treatment methods.