Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.     

Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Continuous production of fructooligosaccharides from sucrose by immobilized fructosyltransferase

The productivity of the continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized onto a high-porous ion exchange resin was optimal with 600 g sucrose/l at a flow rate of 2.7 h^–1 expressed as a superficial space velocity. When the column was operated at 50 °C, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days continuous operation, achieving high productivity of 1174 g/l · h.     

Real-time monitoring of protein secretion in mammalian cell fermentation: measurement of monoclonal antibodies using a computer-controlled HPLC system (BioCad/RPM)

On-line, "real-time" monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG(1), without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. (c) 1995 John Wiley & Sons, Inc.     

Continuous production of fructooligosaccharides using fructosyltransferase immobilized on ion exchange resin

A continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized on a high porous resin, Diaion HPA 25. The optimum pH (5.5) and temperature (55°C) of the enzyme for activity was unaltered by immobilization, and the immobilized enzyme became less sensitive to the pH change. The optimal operation conditions of the immobilized enzyme column for maximizing the productivity were as follows: 600 g/L of sucrose feed concentration, flow rate of superficial space velocity 2.7 h^?1. When the enzyme column was run at 50°C, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days of continuous operation, during which high productivity of 1174 g/L·h was achieved. The kinds of products obtained using the immobilized enzyme were almost the same as those using soluble enzymes or free cells.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Continuous production of l(+)-tartaric acid from cis-epoxysuccinate using a membrane recycle reactor

The one-step bioconversion of cis-epoxysuccinate (CES) to l(+)-tartaric acid by dried Rhodococcus rhodochrous cells containing CES hydrolase activity was studied by using a continuous bioconversion process. The influence of the pH and the temperature was assessed. A mathematical model was used to quantify the CES hydrolase activity and stability. The optimal pH, which resulted in a maximal CES hydrolase activity and stability, was pH 8.0. A large increase in stability (half-life time) could be obtained when the temperature was decreased from 37 to 14°C during the continuous bioconversion. A total bioconversion was maintained for more than 100 days. This resulted in a large value for the specific productivity since the effect of the large increase in stability was much more important than the decrease of activity at the lower temperature. This continuous bioconversion process was further optimised by calculating the productivity for several continuously stirred tank reactors in series. The specific productivity could be nearly doubled when the number of reactors in the series was increased from 1 to 4.     

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.     

Sucrose conversion into palatinose with immobilized Serratia plymuthica cells in a hollow-fibre bioreactor

In recent decades, the production of palatinose has aroused great interest since this structural isomer of sucrose has a promising potential. Using immobilized in a hollow-fibre membrane reactor Serratia plymuthica cells, a complete conversion of concentrated sucrose solutions into palatinose was achieved. Under typical process conditions, the specific productivity of the membrane reactor was 16.8 g m⁻² h⁻¹ (flow rate 1.3 cm³ min⁻¹ and substrate concentration 40%) in continuous mode of action. The activity of the biocatalyst (productivity of the system) decreased slowly with the increase of operation time until the 15th day and remained almost constant to the end of the experiment. The loss of activity was 11% after 90 days of continuous operation. Conversion rate of over 90% was reached for 36–48 h for all concentrations (40–60%) of the substrate solution in cycle mode of action of the bioreactor. The best productivity (18.1 g palatinose m⁻² biocatalytic membrane) for the set period was observed during the recirculation of a 60% sucrose solution. The culture displayed a very good stability under the conditions of critical osmotic stress during the experiments. A microbiological analysis of the end product showed that the produced palatinose syrup measures up to the standards for products of this kind and can be used as an additive to different food products and functional foods.     

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).     

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.     

Lysine production from methanol at 50 degrees C using Bacillus methanolicus: Modeling volume control, lysine concentration, and productivity using a three-phase continuous simulation

A simulation was developed based on experimental data obtained in a 14-L reactor to predict the growth and L-lysine accumulation kinetics, and change in volume of a large-scale (250-m(3)) Bacillus methanolicus methanol-based process. Homoserine auxotrophs of B. methanolicus MGA3 are unique methylotrophs because of the ability to secrete lysine during aerobic growth and threonine starvation at 50 degrees C. Dissolved methanol (100 mM), pH, dissolved oxygen tension (0.063 atm), and threonine levels were controlled to obtain threonine-limited conditions and high-cell density (25 g dry cell weight/L) in a 14-L reactor. As a fed-batch process, the additions of neat methanol (fed on demand), threonine, and other nutrients cause the volume of the fermentation to increase and the final lysine concentration to decrease. In addition, water produced as a result of methanol metabolism contributes to the increase in the volume of the reactor. A three-phase approach was used to predict the rate of change of culture volume based on carbon dioxide production and methanol consumption. This model was used for the evaluation of volume control strategies to optimize lysine productivity. A constant volume reactor process with variable feeding and continuous removal of broth and cells (VF(cstr)) resulted in higher lysine productivity than a fed-batch process without volume control. This model predicts the variation in productivity of lysine with changes in growth and in specific lysine productivity. Simple modifications of the model allows one to investigate other high-lysine-secreting strains with different growth and lysine productivity characteristics. Strain NOA2#13A5-2 which secretes lysine and other end-products were modeled using both growth and non-growth-associated lysine productivity. A modified version of this model was used to simulate the change in culture volume of another L-lysine producing mutant (NOA2#13A52-8A66) with reduced secretion of end-products. The modified simulation indicated that growth-associated production dominates in strain NOA2#13A52-8A66. (c) 1996 John Wiley & Sons, Inc.     

Continuous 2-Keto-l-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia

The fermentation process of 2-keto-L-gulonic acid (2KGA) from L-sorbose was developed using a two-stage continuous fermentation system. The mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium DSM 4026 produced 90 g/L of 2KGA from 120 g/L of L-sorbose at the dilution rate of 0.01 h⁻¹ in a single-stage continuous fermentation process. But after the production period was beyond 150 h, the significant decrease of 2KGA productivity was observed. When the non-spore forming bacteria Xanthomonas maltophilia IFO 12692 was used instead of B. megaterium DSM 4026 as a partner strain for K. vulgare DSM 4025, the 2KGA productivity was significantly improved in a two-stage continuous culture mode, in which two fermentors of the same size and volume were connected in series. In this mode, with two sets of 3-L jar fermentors, the steady state could be continued to over 1,331.5 h at least, when the dilution rates were 0.0382 h⁻¹ and 0.0380 hour⁻¹, respectively, for the first and second fermentors. The overall productivity was calculated to be 2.15 g/L/h at 113.1 g/L and a molar conversion yield of 90.1%. In the up-scaling fermentation to 30-L jar fermentors, 118.5 g/L of 2KGA was produced when dilution rates in both stages were 0.0430 hour⁻¹, and the overall productivity was calculated to be 2.55 g/L/h.     

Mass and thermal balance during composting of a poultry manure—Wood shavings mixture at different aeration rates

Composting is an exothermic process often controlled on the basis of temperature feedback, but for which the energetics of the overall system are generally not well known. In this study, the thermal balance of a poultry manure and wood shavings mixture was estimated during composting at different aeration rates. The study was conducted using 900-l vertical cylindrical reactors at high-flow (0.39–0.52 L/min kg VS) and low-flow (0.07–0.2 L/min kg VS) aeration rates. The actual amount of evaporated water from the high-flow reactor was 37–60% more than from the low-flow reactor in trials 1 and 2. The energy generated from degrading 1 g of VS of the poultry manure and wood shavings mixture was 16.83–19.7 kJ/g VS. The high-flow reactors showed more VS reduction, generating 22–29% more energy than low-flow reactors. The heat loss via forced convection was 52–54% of the total energy produced in the high-flow reactors, but just 17–21% in the low-flow reactors, where natural convection appeared to also play a significant role. The conductive heat loss from the low-flow reactors was greater than from the high-flow reactors, ranging from 44 to 53% in trials 1 and 2. The radiant energy loss was higher in the low-flow reactors, but in all cases was only 5% or less of the total energy loss. Because the reactor scale and configuration will affect these thermal losses, analysis of process energetics should be a fundamental part of system design.     

Seasonal microbial community dynamics in a flowerpot-using personal composting system for disposal of household biowaste

Microbial community dynamics in a flowerpot-using solid biowaste composting (FUSBIC) process were monitored seasonally by quinone profiling and conventional microbiological methods. The FUSBIC system, which consisted of three flowerpots (14 L or 20 L capacity) with 5-6 kg each of a soil-compost mixture (SCM) as the primary reactors, was loaded daily with household biowaste from November 1998 to October 1999. The monthly average waste reduction rate was 88.2% for the 14-L system and 92.5% for the 20-L system on a wet weight basis. The direct total microbial count detected in the 14-L primary reactors ranged from 4.5 to 9.6x10(11) cells.g(-1) of dry wt of SCM, and the viable count of aerobic heterotrophic bacteria recovered on agar plates at 28 degrees C varied from 1.9 to 5.7x10(11) CFU.g(-1) of dry wt. The quinone content of SCM samples from the 14-L and 20-L systems ranged from 160 to 353 nmol.g(-1) of dry SCM. Ubiquinones, unsaturated menaquinones, and partially saturated menaquinones constituted 15.0-36.4, 14.8-22.0, and 41.8-61.6 mol% of the total content, respectively. The major quinone types detected were usually MK-8(H(2)), MK-9(H(2)), and Q-10. Variations in quinone profiles were evaluated numerically by using two parameters, the dissimilarity index (D) and microbial divergence index (MD(q)). The upper limit of seasonal changes in the microbial community structure was about 30% as expressed by D values. The MD(q) values calculated ranged from 18 to 22. A significant positive correlation was found between seasonal temperature and bacterial populations containing partially saturated menaquinones. These results indicated that the FUSBIC system contained highly diverse microbial populations that fluctuated to some extent depending on seasonal temperature. Members of the Actinobacteria were suggested to be the major constituents of the total population present.     

Scale-up of a high cell density continuous culture with Pichia pastoris X-33 for the constitutive expression of rh-chitinase

The feasibility of large-scale production of recombinant human chitinase using a constitutive Pichia pastoris expression system was demonstrated in a 21-L continuous stirred tank reactor. A steady-state recombinant protein concentration of 250 mg/L in the supernatant was sustained for 1 month at a dilution rate of 0.042 h(-1) (equivalent to one volume exchange per day), enabling a volumetric productivity of 144 mg/L d (240 U/L d). The steady-state dry cell weight concentration in this high cell density culture reached 110 g/L. Considering safety and economical aspects, all large-scale cultivations were conducted without molecular oxygen supplementation. Conventional air sparging was used instead. The oxygen demand of the process was determined by off-gas analysis (OUR = 4.8 g O(2) L(-1) h(-1) with k(L)a = 846 h(-1)) and evaluated with regard to further reactor scale-up.     

Optimization of enzymatic gas-phase reactions by increasing the long-term stability of the catalyst

Enzymatic gas-phase reactions are usually performed in continuous reactors, and thus very stable and active catalysts are required to perform such transformations on cost-effective levels. The present work is concerned with the reduction of gaseous acetophenone to enantiomerically pure (R)-1-phenylethanol catalyzed by solid alcohol dehydrogenase from Lactobacillus brevis (LBADH), immobilized onto glass beads. Initially, the catalyst preparation displayed a half-life of 1 day under reaction conditions at 40 degrees C and at a water activity of 0.5. It was shown that the observed decrease in activity is due to a degradation of the enzyme itself (LBADH) and not of the co-immobilized cofactor NADP. By the addition of sucrose to the cell extract before immobilization of the enzyme, the half-life of the catalyst preparation (at 40 degrees C) was increased 40 times. The stabilized catalyst preparation was employed in a continuous gas-phase reactor at different temperatures (25-60 degrees C). At 50 degrees C, a space-time yield of 107 g/L/d was achieved within the first 80 h of continuous reaction.     

Improved β-glucan yield using an Aureobasidium pullulans M-2 mutant strain in a 200-L pilot scale fermentor targeting industrial mass production

β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches are known to be immune activation agents and are incorporated in anti-cancer drugs and health-promoting supplements. β-Glucan concentration was 9.2 g/L in a 200-L pilot scale fermentor using mutant strain Aureobasidium pullulans M-2 from an imperfect fungal strain belonging to A. pullulans M-1. The culture broth of A. pullulans M-2 had a faint yellow color, whereas that of the wild-type had an intense dark green color caused by the accumulation of melanin-like pigments. β-Glucan produced by A. pullulans M-2 was identified as a polysaccharide of D-glucose monomers linked by β-(1→3, 1→6)-glycosidic bonds through GC/MS and NMR analysis. When a conventional medium was used in the culture of A. pullulans M-2 in a 3-L jar fermentor, β-glucan concentration was 1.4-fold that produced by the wild-type. However, when a medium optimized by statistical experimental design was used with dissolved oxygen at 10%, the β-glucan concentration was 9.9 g/L with a yield of 0.52 (g β-glucan/g consumed sucrose), 2.9-fold that of the wild-type. This level of productivity was reproduced when the fermentation was scaled up 200-L. The industrial production of high β-glucan without melanin-like pigments is highly expected, as a health-promoting supplement or functional food.     

Evaluation of an electronic nose for the early detection of organic overload of anaerobic digesters

This study aimed at analysing the utilization of an electronic nose (e-nose) to serve as a specific monitoring tool for anaerobic digestion process, especially for detecting organic overload. An array of non specific metal oxide semiconductor gas sensors were used to detect process faults due to organic overload events in twelve 1.8-L anaerobic semi-continuous reactors. Three different load strategies were followed: (1) a cautious organic load (1.3 gVS L^?1 day^?1); (2) an increasing load strategy (1.3–5.3 gVS L^?1 day^?1) and (3) a cautious organic load with load pulses of up to 12 gVS L^?1 day^?1. A first monitoring campaign was conducted with three different substrates: sucrose, maize oil and a mix of sucrose/oil during 60 days. The second campaign was run with dry sugar beet pulp for 45 days. Hotelling’s T ² value and upper control limit to a reference set of digesters fed with a cautious OLR (1.3 gVS L^?1 day^?1) was used as indirect state variable of the reactors. Overload situations were identified by the e-nose apparatus with Hotelling’s T ² values at least four times higher in magnitude than the upper control limit of 23.7. These results confirmed that the e-nose technology appeared promising for online detection of process imbalances in the domain of anaerobic digestion.