The study of metabolite-separation using an electromicrofiltration appratus

This study was performed to investigate the recovery of dissolved metabolic constituents produced during fermentation, using an electromicrofiltration apparatus. Filtration experiments were performed with crude and with permeate from electromicrofiltration and microfiltration. The results of each method were compared with a focus on measuring the enhancement of the filtrate flux with the application of an electric field. Hydraulic electrofiltration was found to significantly improve filtration and serves as an interesting alternative to cross-flow filtration for the separation of advantagenous constituents, such as amino acids and biopolymers from the fermentation broth.     

The Effect of Gas Sparging in Cross‐Flow Microfiltration of 2,3‐Butanediol Fermentation Broth

Recovery of 2,3‐butanediol from a fermentation broth entails the separation of cells and other suspended solids as the initial step for subsequent separation stages. The aim of this work was to study the cross‐flow filtration of broth in the fermentation of 2,3‐butanediol from blackstrap molasses by Klebsiella oxytoca (NRRL B‐199). A plate type laboratory scale cross‐flow microfiltration unit with a 0.2‐μm cellulose acetate membrane was employed for this purpose. Preliminary results showed that the permeate flux would decline rapidly due to fouling caused by the natural impurities of blackstrap molasses, and modifications of the conventional cross‐flow filtration would be essential to achieve a filtration rate appropriate for practical purposes. In this work, the permeate flux was enhanced by air sparging, which scoured the membrane surface of colloidal deposits and allowed a practical filtration rate to be maintained. The average permeate flux increased by 39 % and 54 % for an air sparging rate of 0.5 L/min and 1.0 L/min respectively, in the case of an initial biomass concentration of 4.66 g/L. For an initial biomass concentration of 14.2 g/L, the flux increased by 105 % and 146 % for the gas rate of 0.5 and 1.0 L/min, respectively. It may be concluded that gas sparging is beneficial in cross‐flow filtration of thick suspensions like a fermentation broth.     

Recovery of alkaline proteases by membrane filtration

This work describes the recovery of an extracellular alkaline protease from fermentation broths of a Bacillus sp ATCC 21536, at pH=10.0 using ultrafiltration (MWCO 100,000) and microfiltration (0.1 m) membranes in hollow fiber devices. The influence of membrane pore size and polymeric material and membrane filtration performance was studied. High protein recoveries and high average flux rates were obtained with polysulfone membranes. A decrease of concentration polarization was obtained, simultaneously with enhancement of filtration flux rate and enzyme recovery by using submicron sized charged particles. These polymers lead to flocculation and adsorption of whole cells and soluble factors from the fermentation broth. The best results were obtaiend by combination of cationic (0.1%) and anionic (0.04%) polymers.     

微滤膜分离提纯苦楝素的研究

通过对不同孔径和材质的微孔滤膜对苦楝提取液过滤分离比较,优选出孔径为0.45μm的聚醚砜微滤膜对苦楝提取液具有良好的过滤性能。确定的膜分离提纯苦楝素优化工艺条件是:在料液浓度为0.374 mg/mL,料液温度35℃,操作压力差为0.08 MPa,循环流量为0.15 L/h,pH=7.0,苦楝素的转移率为99.4%,除杂率为8.3%,通量为147.2 L/m2.h,苦楝素的纯度为由提取液的0.89%,提高到了8.79%。     

Studies on the interaction of fermentation and microfiltration operations: erythromycin recovery from Saccharopolyspora erythraea fermentation broths

Changes in fermentation media not only affect the performance of the fermentation itself (with regard to the kinetics of biomass and product formation and the yields obtained) but also the initial product-recovery operations downstream of the fermentor. In this work, microfiltration experiments to remove Saccharopolyspora erythraea biomass from fermentation broth and to recover erythromycin were carried out using two fundamentally different media; a soluble complex medium (SCM) and an oil-based process medium (OBM). Small-scale batch fermentations of 14-L working volume were carried out in triplicate using both media. Broth samples were taken from each fermentation at regular intervals from the end of the exponential-growth phase onwards. These were then processed using a Minitan II (acrylic), tangential crossflow-filtration module, fitted with a single 60 cm(2) Durapore hydrophilic 0.2 microm membrane, operated in concentration mode. The OBM fermentations produced higher titers of erythromycin but required longer fermentation times due to increased lag phases and slower maximum-growth rates. The OBM also increased the loading on the membrane; at maximum product titers residual oil concentrations of 3 g. L(-1), antifoam concentrations of 2 g. L(-1) and flour concentrations estimated at approximately 10 g/L(-1) were typical. It was found that both the permeate flux and erythromycin transmission were affected by the choice of medium. The OBM had significantly lower values for both parameters (12.8 Lm(-2) h(-1) and 89.6% respectively) than the SCM (35.9 Lm(-2) h(-1) and 96.7% respectively) when the fermentations were harvested at maximum erythromycin titers. Transmission of erythromycin stayed approximately constant as a function of fermentation time for both media, however, for the OBM the permeate flux decreased with time which correlated with an increase in broth viscosity. The relatively poor microfiltration performance of the OBM medium was, however, offset by the higher titers of erythromycin that were achieved during the fermentation. The filtration characteristics of the SCM broth did not show any correlation with either broth viscosity or fermentation time. Image-analysis data suggested that there was a correlation between hyphal morphology (main hyphal length) and permeate flux (no such correlation was found for the OBM broth). Moreover, it has been shown for the OBM broth that the residual flour had a profound effect on the microfiltration characteristics. The influence of the residual flour was greater than that imposed by the morphology and concentration of the biomass. The understanding of the factors governing the interaction of the fermentation and microfiltration operations obtained in this work provides a first step towards optimization of the overall process sequence.     

Production and separation of alpha-agarase from Altermonas agarlyticus strain GJ1B

This paper presents results on the production of alpha-agarase by a fermentation process and its separation using membrane microfiltration (MF). Optimization of fermentation conditions for alpha-agarase production using Altermonas agarlyticus grown on medium containing agar as a carbon source was done in batch, fed-batch and continuous fermentations. Continuous culture at a dilution rate of 0.03 h(-1) appeared to be best suited for production of alpha-agarase by this organism. At 0.03 h(-1) dilution rate, enzyme activity was 0.9 U/ml. Clarification of broth was done using a hollow-fibre microfiltration membrane. The influence of hydrodynamic parameters on permeate flux and enzyme activity was studied. The best performance was obtained with prefiltered fermentation broth. A stable permeate flux of about 250-270 ml/min.m2 and an enzyme retention rate between 0% and 25% was obtained at temperatures between 6 degrees C and 22 degrees C, transmembrane pressure of 100 mm Hg and fluid cross-flow velocity of 4 x 10(-2) m/s. From the experiments on concentration of fermentation broth, the best compromise between enzyme activity transmission and permeate flux was obtained at a concentration factor of 2.     

A new integrated membrane filtration and chromatographic device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.     

Effectiveness of Biosorption-Assisted Microfiltration Process for Treatment of Domestic Wastewater

The efficiency of a biosorbent prepared from Eichhornia crassipes roots (ECR) was explored for the treatment of domestic sewage water in combination with low-cost ceramic microfiltration membrane. Batch sorption studies were conducted as a function of biosorbent dose, initial chemical oxygen demand (COD) loading, and temperature. Sorption equilibrium data of varying initial COD values (116–800 mg/L) indicated high potential of ECR for COD removal. Using 0.25 g/L of biosorbent dose, the equilibrium adsorption capacity was obtained as 2480 mg/g at 20°C for an initial COD loading of 800 mg/L. Microfiltration study was performed using ceramic membrane made from composition of α-alumina and clay. The effect of operating parameters on filtration characteristics was observed in terms of permeate flux. Permeate samples were characterized in terms of various parameters both for the direct filtration, as well as biosorbent-assisted filtration. The filtration behavior of wastewater at varying transmembrane pressure was explained using various membrane fouling models. The results suggested that microfiltration of domestic wastewater with incorporation of biosorbent (0.25 g/L) was highly effective for removal of organic load (>90%), turbidity (>99%), and total suspended solids (TSS) (93–95%) and the treated water quality was suitable for reuse in various purposes, such as gardening, floor and car washing, etc.     

Flux enhancement for membrane filtration of bacterial suspensions using high-frequency backpulsing

A promising method for reducing membrane fouling during crossflow microfiltration of biological suspensions is backpulsing. Very short backpulses (0.1-1.0 s) have been used to increase the net flux for washed bacterial suspensions and whole bacterial fermentation broths. The net fluxes under optimum backpulsing conditions for the washed bacteria are approximately 10-fold higher than those obtained during normal crossflow microfiltration operation, whereas only a 2-fold improvement in the net flux is achieved for the fermentation broths. A theory is presented that is based on external fouling during forward filtration and nonuniform cleaning of the membrane during reverse filtration. The model contains an adjustable parameter which is a measure of the cleaning efficiency during backpulsing; the cleaning efficiency found by fitting the model to the experiments increases with increasing frequency and duration of the backpulses. The theory predicts an optimum backpulsing frequency, as was observed experimentally. An economic analysis shows that crossflow microfiltration with backpulsing has lower costs than centrifugation, rotary vacuum filtration, and crossflow microfiltration without backpulsing.     

Development of crossflow filtration processes for the commercial-scale isolation of a bacterial lipase

Isolation of a lipase produced by Pseudomonas aeruginosa (MW 29,000) employed crossflow microfiltration for production of a cell-free enzyme solution and crossflow ultrafiltration for concentration of the enzyme and removal of low molecular weight impurities. Poor flux and enzyme permeation were measured during initial screening of various microfiltration membrane types for isolation of the enzyme from a peptonized-milk-based broth; the results suggested that a soluble broth component was forming a gel layer which controlled both hydraulic flux and enzyme permeation. Reformulation of the fermentation medium resulted in enhanced performance, obtaining fluxes of 40 l/h m² and enzyme permeation of 50% on hydrophilically-modified PVDF membranes and resulted in a feasible clarification process. Enzyme permeation remained constant with respect to activity in the feed, rather than being proportional to activity in the retentate; it was hypothesized that this resulted from a concomitant concentration of the gel-forming components with cell concentration. Concentration of the clarified enzyme solution was performed using 30 000 MWCO regenerated cellulose membranes. Complete enzyme retention and high flux (57 l/h m²) were maintained through a 130-fold concentration of the microfiltrate. As both systems were taken to the 100 and 1000 l scales, similar filtration performances were obtained with system hold-up volume and pump cavitation becoming important considerations at the larger scales. Excellent reproducibility was observed in a series of eight large-scale experiments.     

Factors affecting membrane fouling in filtration of Saccharomyces cerevisiae in an internal ceramic filter bioreactor

Filtration of ethanol fermentation medium and broth by using symmetric and asymmetric ceramic membranes has been studied in an internal filter bioreactor. Factors studied included membrane structure and pore size, medium sterilization, and concentrations of glucose, yeast extract in the medium, yeast cell and protein in broth. The aim was to determine the main factors responsible for the decline in filtration performance during ethanol fermentation by Saccharomyces cerevisiae. Flux index (Fi) of a new concept has been developed to evaluate the degree of flux decline during the membrane fouling process. Fi was defined as the ratio of the membrane flux at certain filtration time (t?=?t) to the initial (t?=??0) flux of pure water, not the initial (t?=?+0) flux of the test fluid. Flux with sterilized medium was approximately two-fold higher than that with unsterilized medium although the reason could not be explained clearly. Glucose, interaction between glucose and yeast extract, yeast cells, and proteins in fermentation broth were found to play an important part in membrane fouling. Fi of the symmetric membrane decreased to a less extent than that of the asymmetric membrane with increasing glucose concentration. But, the result with various yeast cell concentrations turned out to be contrary. Fouling was more serious for asymmetric membrane during the filtration of fermentation supernatant. This was thought to be due to different fouling mechanisms for the two types of membrane.     

Microfiltration of streptococcal fermentation broths: study of the factors affecting the concentration effect

Streptokinase (SK) recovery from streptococcal fermentation broth by cross-flow microfiltration has been studied. Recovery of SK in the filtrate, independent of the volumetric concentration factor, is approximately two-fold lower than the initial SK activity in the fermentation broth; moreover, the SK activity in the retentate increase during the process, reaching a concentration factor of 2.73. These results show that the membrane works more as an ultrafiltration membrane, with rejection of S = 0.6, than as a microfiltration membrane. Under filtration conditions, the membrane permeation rate decreased with time. This decreased could be explained by deposition and interaction of material onto/with the membrane resulting in the concentration of permeable products. Studies of the individual concentration factors for the main streptococcal exocellular proteins, indicate clearly that the concentration of the proteins during the microfiltration process is independent of the size of the proteins, suggesting that other factors, such as charge and hydrophobicity, along with concentration-polarization, should be taken also into account for the understanding of this phenomenon. (c) 1994 John Wiley & Sons, Inc.     

Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.     

Removal of organics and ions from wastewater using a hybrid system of microfiltration membrane and microorganisms

Microfiltration membranes are not able to remove organic compounds. A hybrid system of a microfiltration membrane and bacteria was designed for separation of organic compounds and ions from wastewater. Colonies of bacteria (Escherichia coli) were subjected to sedimentation on the surface of a microfiltration membrane (0.2 μm cellulose acetate) as a complementary part of the system to enhance the removal efficiency. Three selected categories of materials i.e. preservative substances, cephalosporins and ions were used to prepare the synthetic feed. The results indicate that preservatives were reduced more than 80%, cephalosporins were removed around 60% and ions decreased more than 50% in the feed solution using this hybrid system for filtration. The interaction between bacteria and chemical materials is responsible for removal of organic compounds and ions from test solutions. However the interaction decreased over time due to the limited capacity of the bacteria.     

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 microm ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations.     

Crossflow filtration of yeast broth cultivated in molasses

A broth of yeast cells cultivated in molasses was crossfiltered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mum). The steady-state flux was one-twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mum in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 mum, but using larger pores of 3 to 5 mum it returned almost to the bases line. (c) 1994 John Wiley & Sons, Inc.     

Fouling effects of yeast culture with antifoam agents on microfilters

The fouling effects of yeast fermentation broths of Candida utilis in the presence of various commercial antifoam agents (PPG2000, B5600, and G832) up to 4.0 mL/L were studied, using Millipore polyvinylidene fluoride 0.22-mum hydrophilic membranes (GVWP), in a stirred-cell system at 50 kPa and 700 rpm. PPG2000, which has a low value of work of adhesion (W(a) of 0.81 mN/m), gave a steady flux of broth of 29 L/(h m(2)) and was found to have no significant fouling effect on the microfiltration of broth. G832, which has a high W(a), (26.0 mN/m) reduced the flux of the broth to 17 L/(h m(2)); i.e., by 42% when only 1.0 mL/L was used. However, B5600, which has a W(a) of 14.3 mN/m, was found to enhance the flux of broth to 54 L/(h m(2)); i.e., by 86%, due to the preferential adsorption of the B5600 components onto the hydrophobic cell contents released. These results were reinforced by the depressurization experiments performed with both hydrophilic (GVWP) and hydrophobic (GVHP) membranes, using both young and aged broths. B5600 was found to be the optimum antifoam agent in this study in terms of membrane performance and defoaming efficiency. (c) 1997 John Wiley & Sons, Inc.     

Filtration kinetics of chitosan separation by electrofiltration

Downstream processing of chitosan requires several technological steps that contribute to the total production costs. Precipitation and especially evaporation are energy-consuming processes, resulting in higher costs and limiting industrial scale production. This study investigated the filtration kinetics of chitosan derived from cell walls of fungi and from exoskeletons of arthropods by electrofiltration, an alternative method, thus reducing the downstream processing steps and costs. Experiments with different voltages and pressures were conducted in order to demonstrate the effect of both parameters on filtration kinetics. The concentration of the biopolymer was obtained by the average factor of 40 by applying an electric field of 4 V/mm and pressure of 4 bars. A series of analytical experiments demonstrated the lack of structural and functional changes in chitosan molecules after electrofiltration. These results, combined with the reduction of energy and processing time, define the investigated method as a promising downstream step in the chitosan production technology.     

Microfiltration of yeast suspensions with self-cleaning spiral vortices: possibilities for a new membrane module design

A novel method of producing controlled vortices was used to reduce both concentration polarization and membrane fouling during microfiltration of Saccharomyces cerevisiae broth suspensions. The method involves flow around a curved channel at a sufficient rate so as to produce centrifugal instabilities (called Dean vortices). These vortices depolarize the build-up of suspended particles such as yeast cells at the membrane-solution interface and allow for increased membrane permeation rates. Various operating conditions under which such vortices effectively reduced cake build-up of suspended particles such as yeast cells at the membrane-solution interface and allow for increased membrane permeation rates. Various operating conditions under which such vortices effectively reduced cake build-up during microfiltration of 0 to 0.55 dry wt% yeast broth were investigated. Flux improvements of over 60% for 0.25 dry wt% yeast broth for flow with over that without Dean vortices were observed. This beneficial effect increased with increasing retentate flow rate and increasing transmembrane pressure and decreased with increasing concentration of suspended matter. Similar behavior was observed whether the cells were viable of killed. the improvement in flux in the presence over that in the absence of vortices correlated well with centrifugal force or azimuthal velocity squared. The relative cake resistances increased with reservoir yeast concentration. These values with vortices increased from 62% to 75% of that without vortices with increasing yeast concentration. The ratio of the cake thicknesses in the limiting case (at high feed concentration) was 3.25. These results suggest that self-cleaning spiral vortices could be effective in maintaining good and steady microfiltration performance with cell suspensions other than those tested. (c) 1995 John Wiley & Sons, Inc.     

Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation.

A silicone membrane was used to study butanol separation from model butanol solutions and fermentation broth. Depending upon the butanol feed concentration in the model solution and pervaporation conditions, butanol selectivities of 20.88-68.32 and flux values of 158.7-215.4 g m(-)(2) h(-)(1) were achieved. Higher flux values (400 g m(-)(2) h(-)(1)) were obtained at higher butanol concentrations using air as sweep gas. In an integrated process of butanol fermentation-recovery, solvent productivities were improved to 200% of the control batch fermentation productivities. In a batch reactor the hyper-butanol-producing mutant strain C. beijerinckii BA101 utilized 57.3 g/L glucose and produced 24.2 g/L total solvents, while in the integrated process it produced 51.5 g/L (culture volume) total solvents. Concentrated glucose medium was also fermented. The C. beijerinckii BA101 mutant strain was not negatively affected by the pervaporative conditions. In the integrated experiment, acids were not produced. With the active fermentation broth, butanol selectivity was reduced by a factor of 2-3. However, the membrane flux was not affected by the active fermentation broth. The butanol permeate concentration ranged from 26.4 to 95.4 g/L, depending upon butanol concentration in the fermentation broth. Since the permeate of most membranes contains acetone, butanol, and ethanol (and small concentrations of acids), it is suggested that distillation be used for further purification.