Performance of cross-linked enzyme crystals of engineered halohydrin dehalogenase HheG in different chemical reactor systems

Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β-substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross-linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross-linked with bis-maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space-time yield of 23.9 kg_product g_CLEC⁻¹ h⁻¹ L_{reactor volume}⁻¹ along with the highest achieved product enantiomeric excess [64%] for application in a packed-bed reactor. Additionally, lyophilization of those CLECs yielded a storage-stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature.     

Continuous adsorption and recovery of Cr(VI) in different types of reactors

This study reports the results of experiments on continuous adsorption and desorption of Cr(VI) ions by a chemically modified and polysulfone-immobilized biomass of the fungus Rhizopus nigricans. A fixed quantity of polymer-entrapped biomass beads corresponding to 2 g of dry biomass powder was employed in packed bed, fluidized bed, and stirred tank reactor for monitoring the continuous removal and recovery of Cr(VI) ions from aqueous solution and synthetic chrome plating effluent. Parameters such as flow rate (5, 10 and 15 mL/min), inlet concentration of Cr(VI) ions (50, 100, 150 and 250 mg/L) and the depth of biosorbent packing (22.8, 11.2 and 4.9 cm) were evaluated for the packed bed reactor. The breakthrough time and the adsorption rates in the packed bed column were found to decrease with increasing flow rate and higher Cr inlet concentrations and to increase with higher depths of sorbent packing. To have a comparative analysis of Cr adsorption efficiency in different types of reactors, the fluidized bed reactor and stirred tank reactor were operated using the same quantities of biosorbent material. For the fluidized bed reactor, Cr(VI) solution of 100 mg/L was pumped at 5 mL/min and fluidized by compressed air at a flow rate of 0.5 kg/cm.(2) The stirred tank reactor had a working volume of 200 mL capacity and the inlet/outlet flow rate was 5 mL/min. The maximum removal efficiency (mg Cr/g biomass) was obtained for the stirred tank reactor (159.26), followed by the fluidized reactor (153.04) and packed bed reactor (123.33). In comparison to the adsorption rate from pure chromate solution, approximately 16% reduction was monitored for synthetic chrome plating effluent in the packed bed. Continuous desorption of bound Cr ions from the reactors was effective with 0.01 N Na(2)CO(3) and nearly 80-94% recoveries have been obtained for all the reactors.     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l^-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: c_Glc – glucose concentration, mmol l^-1; c_Gln – glutamine concentration, mmol l^-1; c_Amm – ammonia concentration, mmol l^-1; c_Lac – lactate concentration, mmol l^-1; c_MAb – MAb concentration, mg l^-1; D – dilution rate, d^-1; D_i – dilution rate in the inner chamber of the membrane dialysis reactor, d^-1; D₀ – dilution rate in the outer chamber of the membrane dialysis reactor, d^-1; q*_FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1; q*_FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1.     

Conversion of cellulose into fungal cell mass in solid state culture

Summary To satisfy the demand for simple production technology (simple and cheap reactor, cheap recovery and finishing), solid state cultivations were carried out with pretreated straw in a simple fixed bed reactor under nonsterile conditions.The results of these investigations were compared with those evaluated in a stirred tank reactor. The same cell mass fractions were obtained in both reactors. However, about double the cultivation time is necessary for a solid state cultivation as compared to a submerse cultivation.Symbols N₂ nitrogen content of dry biomass (%) - P productivity on cell protein (%/h) - T temperature (°C) - t_F cultivation time (h) - X fungal cell mass fraction (%)     

Cadmium uptake by algal biomass in batch and continuous (CSTR and packed bed column) adsorbers

In this work, the properties of marine algae Gelidium, algal waste from agar extraction industry and a composite material were investigated for cadmium(II) biosorption. Equilibrium experiments were performed at three pH values (4, 5.3 and 6.5). Equilibrium data were well described by the Langmuir and Langmuir–Freundlich isotherms. Two models predicting the pH influence in the cadmium biosorption (discrete and continuous models) have been developed in order to better describe the equilibrium. The continuous model also considers a heterogeneous distribution of carboxylic groups, determined by potentiometric titration. The results of batch kinetic experiments performed at different pH values were well fitted by two mass transfer models and the homogeneous diffusion coefficients for the cadmium ions inside the biosorbent were obtained. Continuous stirred tank reactor (CSTR) and packed bed column configurations were also examined for the biosorption of cadmium ions. A strong acid (0.1 M HNO₃) was used as eluant to regenerate the biosorbents in the column. Several mass transfer models were applied with success to describe the biosorption process in batch mode, CSTR and fixed bed column.     

Test of Porous Ceramic Material for the Immobilisation of Predominant Nitrifying Bacteria and for the Improvement of the AAO Process

The efficiency of nitrification in a fixed bed reactor was compared to that found in an activated sludge reactor to determine their sensitivity to changing loads and lower temperatures. Two structurally identical lab‐scale systems, using the anaerobic/anoxic/aerobic (AAO) process to remove nitrogen and phosphorus simultaneously, were operated in parallel with secondary clarifiers and sludge return. The first aerobic system was operated as an activated sludge reactor, the second system as a fixed bed reactor. The aerobic fixed bed reactor was filled with porous ceramic materials for the immobilisation of predominantly nitrifying bacteria. The removal efficiencies of 99 % NH₄⁺‐N, 90 % DOC, and 98 % PO₄^3–‐P for normal loads of 0.11 g/L d DOC, 0.06 g/L d NH₄⁺‐N, and 0.0054 g/L d PO₄^3–‐P were achieved for both systems. However, the system with an aerobic fixed bed reactor was characterised by the following advantages over the system with an activated sludge reactor: a shorter time to reach almost complete nitrification, a higher nitrification rate at higher loads of NH₄⁺‐N and a lower sensitivity of nitrification at lower temperatures down to 12 °C.     

Purification of exhaust air containing organic pollutants in a trickle-bed bioreactor

Summary This paper reports experiments on the purification of exhaust air containing organic pollutants by a new biological process using a trickle-bed reactor. Pollutant-specific microorganisms in high concentration were fixed to a suitable bed. The absorption and conversion of propionaldehyde as a model pollutant was measured by systematic variation of the gas and liquid flow rates in the reaction system. At a space velocity of 1000 h^–1, it was possible to achieve conversion rates of between 68 and 96%, depending on the trickling density. The degradation capacity of the biological trickle bed is over 500 g propionaldehyde/m³ of reactor per hour. By using a tube bundle (honeycomb tube), it was possible to ensure continuous operation of the reactor with reduced conversion and pressure loss.Dedicated to Professor Dr. Dr. D. Behrens on the occasion of 65th birthday     

Biofiltration of Butyl Acetate and Xylene Mixtures Using a Trickle‐Bed Air Biofilter

Butyl acetate and xylene mixtures are commonly encountered from the manufacture of semi‐conductor or opto‐electronic apparatuses. The release of these substances into the ambient air may have a negative effect on the air quality. This study attempts to employ a trickle‐bed air biofilter for treating butyl acetate and xylene mixtures under different gas flow rates and influent concentrations. Almost complete VOC removal could be attained with influent carbon loadings of BA (butyl acetate) and X (xylene) below 40 and 15 g/m³h, respectively. As the influent carbon loadings of BA and X were increased up to 150 and 110 g/m³h, removal efficiencies higher than 80 % were achieved. Therefore, the trickle‐bed air biofilter (TBAB) appeared efficient in the control of emissions containing mixtures of butyl acetate and xylene with low to medium carbon loadings. The removal efficiencies of butyl acetate were higher than those of xylene, indicating that butyl acetate was the substrate preferred in the utilization of butyl acetate and xylene mixtures by the microorganisms. Carbon recoveries of 98–101 % were achieved, demonstrating the accuracy of results. The carbon mass rate of the liquid effluent was approximately two to three orders of magnitude less than that of the CO₂ effluent, indicating that the dissolved VOCs and their derivatives in the leachate were present in a negligible amount in the reactor. Applicable operating conditions of the TBAB unit for treating BA and X mixtures were suggested.     

Dyestuff wastewater treatment using chemical oxidation,physical adsorption and fixed bed biofilm process

Fenton’s oxidation and activated carbon adsorption were examined as pretreatment processes for dyestuff wastewater having high salinity, colour, and non-biodegradable organic concentrations. In this work, each wastewater stream produced by individual production processes was classified as streams R1, R2, and R3. The stream having a value of BOD₅/COD lower than 0.4 was pretreated by Fenton’s oxidation or activated carbon adsorption to increase the ratio of BOD₅/COD which indicates biodegradability. For Fenton’s oxidation with one stream having a value of BOD₅/COD lower than 0.4, the optimal reaction pH was 3.0 and the minimum dosing concentration (mg l⁻¹) of H₂O₂:FeSO₄·7H₂O was 700:3500. Stream R3, which consisted mainly of methanol was efficiently treated by activated carbon adsorption. The ratio of BOD₅/COD was also increased to 0.432 and 0.31 from 0.06 in Fenton’s oxidation and activated carbon adsorption, respectively. A biological treatment system using a fixed bed reactor was also investigated to enhance biological treatment efficiency at various hydraulic retention times, pretreatment conditions by Fenton’s reagent and salt concentrations by dyestuff wastewater. In addition, the efficiency of Fenton’s oxidation as a post-treatment system was also investigated to present a total treatment process of dyestuff wastewater. As the influent COD and salinity were increased, the effluent SS and COD were consequently increased. However, as the microorganisms became adapted to the changed influent condition, the treatment efficiency of the fixed bed reactor quickly recovered under the high COD and salinity since the microorganisms were well adapted to toxic influent conditions. A wastewater treatment process consisting of chemical oxidation, activated carbon adsorption, fixed bed biofilm process and Fenton’s oxidation as a post-treatment system can be useful to treat dyestuff wastewater having high salinity, colour, and non-biodegradable organic concentration.     

Utilizing the tubular bioreactor for continuous recovery of secreted fusion protein from recombinant Escherichia coli

In order to improve the cultivation properties of a traditional continuous stirred tank reactor (CSTR), we introduced a circulation unit made of four inorganic membranes in stainless steel tubes in parallel configuration, the so-called Tubular Bioreactor (TBR). Furthermore, the TBR outlet tube, which has a restriction nozzle at the end, was installed on top of the fermentor vessel, thereby creating a strong jet flow into the reactor and thus improving the mixing and the oxygen transfer rate. The k _La could be increased by approximately 50%. This setup was used for cultivations of recombinant Escherichia coli in a minimal medium and high cell density. More than 50 g dry cell mass/dm³ was obtained. Simultaneously, we have produced an elongated form of human insulin-like growth factor II, which was a secreted fusion protein utilizing the E. coli secretion system based on staphylococcus protein A. The product could be recovered continuously through the TBR-membrane.     

Nitrogen removal performance of a hybrid anammox reactor

The anaerobic ammonium oxidation (anammox) process has attracted considerable attention in recent years as an alternative to conventional nitrogen removal technologies. In this study, an innovative hybrid reactor combining fluidized and fixed beds for anammox treatment was developed. The fluidized bed was mechanically stirred and the gaseous product could be rapidly released from the anammox sludge to prevent washout of the sludge caused by floatation. The fixed bed comprising a non-woven biomass carrier could efficiently catch sludge to reduce washout. During the operation, nitrogen loading rates to the reactor were increased to 27.3 kg N/m³/d, with total nitrogen removal efficiencies of 75%. The biomass concentration in the fluidized bed reached 26-g VSS/L. Anammox granules were observed in the reactors, with settling velocities and sludge volumetric index of 27.3 ± 6.5 m/h and 23 mL/g, respectively. Quantification of extracellular polymeric substances revealed the anammox granules contained a significant amount of extracellular proteins.     

Ethanol production in an immobilized-cell reactor

Biomass can be converted to sugars by hydrolysis with enzymes or mineral acids. These sugars can be converted into a number of chemical intermediates in biological reactors. Biological reactions are generally slow and selection of the most efficient reactor is important in these applications. Immobilized-cell reactors allow high cell densities and high throughput by attaching the microorganisms to a fixed support. This paper examines the rate of production of ethanol from glucose by Saccharomyces cerevisia in a packed column. These rates are compared with those for the same reaction in a stirred reactor.     

Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii

Citrobacter freundii DSM 30040 immobilized on modified polyurethane carrier particles PUR 90/16 was used for continuous glycerol fermentation in an anaerobic fixed bed reactor with effluent recycle and pH control (fixed bed loop reactor). The fermentor was run with buffered mineral medium under growth conditions resulting in the permanent renewal of active biomass. The effects of glycerol concentration in the feed, dilution rate (D), pH and temperature (T) were investigated to optimize the process. With 400 mm glycerol in the feed, pH 6.9, T = 36°C and D = 0.5 h^–1 the maximum productivity could be determined as 8.2 g/l per hour of 1,3-propanediol.     

Detailed Assessment of the Kinetics of Hg-Cell Association,Hg Methylation,and Methylmercury Degradation in Several Desulfovibrio Species

The kinetics of inorganic Hg [Hg(II)_i] association, methylation, and methylmercury (MeHg) demethylation were examined for a group of Desulfovibrio species with and without MeHg production capability. We employed a detailed method for assessing MeHg production in cultures, including careful control of medium chemistry, cell density, and growth phase, plus mass balance of Hg(II)_i and MeHg during the assays. We tested the hypothesis that differences in Hg(II)_i sorption and/or uptake rates drive observed differences in methylation rates among Desulfovibrio species. Hg(II)_i associated rapidly and with high affinity to both methylating and nonmethylating species. MeHg production by Hg-methylating strains was rapid, plateauing after ∼3 h. All MeHg produced was rapidly exported. We also tested the idea that all Desulfovibrio species are capable of Hg(II)_i methylation but that rapid demethylation masks its production, but we found this was not the case. Therefore, the underlying reason why MeHg production capability is not universal in the Desulfovibrio is not differences in Hg affinity for cells nor differences in the ability of strains to degrade MeHg. However, Hg methylation rates varied substantially between Hg-methylating Desulfovibrio species even in these controlled experiments and after normalization to cell density. Thus, biological differences may drive cross-species differences in Hg methylation rates. As part of this study, we identified four new Hg methylators (Desulfovibrio aespoeensis, D. alkalitolerans, D. psychrotolerans, and D. sulfodismutans) and four nonmethylating species (Desulfovibrio alcoholivorans, D. tunisiensis, D. carbinoliphilus, and D. piger) in our ongoing effort to generate a library of strains for Hg methylation genomics.     

Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered β-hexosaminidase immobilized on solid support

The D746E variant of Bifidobacterium bifidum β-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective β-1,3-glycosylation of lactose from a d -glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145–200 mM) at quantitative yield from the donor substrate. The wild-type β-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d -glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80–100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.     

Starter culture production in fluidized bed reactor with a flocculent strain ofL. plantarum

Summary A lactic starter culture of a flocculentLactobacillus plantarum was produced in a fluidized bed reactor with higher cell volumetric productivities than in a continuous stirred tank reactor. The fluidized bed reactor was operated at optimised parameters obtained in batch reactor performed with and without pH control.     

Biodegradation of phenol and chlorophenols with defined mixed culture in shake-flasks and a packed bed reactor

Pseudomonas testosteroni CPW301 degraded phenol and 4-chlorophenol simultaneously, but degradation rates of these compounds were affected by 4-chlorophenol. Phenol increased the cell concentration and therefore the degradation efficiency of 4-chlorophenol was improved. Pseudomonas solanacearum TCP114 could degrade only 2,4,6-trichlorophenol. A defined mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 could treat phenol, 4-chlorophenol, and 2,4,6-trichlorophenol completely and overcome the inhibition of substrates to other microorganisms. The degradation capacity of the packed bed reactor (PBR) was higher than that of the continuous stirred tank reactor, but the PBR was unsuitable for oxygen-sensitive microorganisms.     

Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates

This article describes the use of biofilm reactors for the production of various chemicals by fermentation and wastewater treatment. Biofilm formation is a natural process where microbial cells attach to the support (adsorbent) or form flocs/aggregates (also called granules) without use of chemicals and form thick layers of cells known as "biofilms." As a result of biofilm formation, cell densities in the reactor increase and cell concentrations as high as 74 gL^-1 can be achieved. The reactor configurations can be as simple as a batch reactor, continuous stirred tank reactor (CSTR), packed bed reactor (PBR), fluidized bed reactor (FBR), airlift reactor (ALR), upflow anaerobic sludge blanket (UASB) reactor, or any other suitable configuration. In UASB granular biofilm particles are used. This article demonstrates that reactor productivities in these reactors have been superior to any other reactor types. This article describes production of ethanol, butanol, lactic acid, acetic acid/vinegar, succinic acid, and fumaric acid in addition to wastewater treatment in the biofilm reactors. As the title suggests, biofilm reactors have high potential to be employed in biotechnology/bioconversion industry for viable economic reasons. In this article, various reactor types have been compared for the above bioconversion processes.     

Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of Qs_rAb to .Abbreviations C concentration - D dilution rate - FBR fixed bed bioreactor - FIBR fluidised bed bioreactor - Gln glutamine - Qs cell specific rate - Qv volumetric rate - rAb recombinant antibody - X_v viable cell density - specific growth rate