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.     

Separation of lactic acid-producing bacteria from fermentation broth using a ceramic microfiltration membrane with constant permeate flow

The influence of several operating parameters on the critical flux in the separation of lactic acid-producing bacteria from fermentation broth was studied using a ceramic microfiltration membrane equipped with a permeate pump. The operating parameters studied were crossflow velocity over the membrane, bacterial cell concentration, protein concentration, and pH. The influence of the isoelectric point (IEP) of the membrane was also investigated. In the interval studied (5.3-10.8 m/s), the crossflow velocity had a marked effect on the critical flux. When the crossflow velocity was increased the critical flux also increased. The bacterial cells were retained by the membrane and the concentration of bacterial cells did not affect the critical flux in the interval studied (1.1-3.1 g/L). The critical flux decreased when the protein concentration was increased. It was found that the protein was adsorbed on the membrane surface and protein retention occurred even though the conditions were such that no filter cake was present on the membrane surface. When the pH of the medium was lowered from 6 to 5 (and then further to 4) the critical flux decreased from 76 L/m(2)h to zero at both pH 5 and pH 4. This was found to be due to the fact that the lowering in pH had affected the physiology of the bacterial cells so that the bacteria tended to adhere to the membrane and to each other. The critical flux, for wheat flour hydrolysate without particles, was much lower (28 L/m(2)h) when using a membrane with an IEP of 5.5 than the critical flux of a membrane with an IEP at pH 7 (96 L/m(2)h). This was found to be due to an increased affinity of the bacteria for the membrane with the lower IEP.     

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.     

Microfiltration and ultrafiltration of polysaccharides produced by fermentation using a rotating disk dynamic filtration system

The recovery of exopolysaccharides (EPS) produced by Sinorhizobium meliloti bacteria by dynamic microfiltration was investigated using a rotating disk device designed in our laboratory, equipped with a 0.2 microm nylon membrane. This system differs from commercially available systems by the presence of vanes on the disk which produce a very important increase in permeate flux while yielding excellent EPS transmission. For polymers produced under standard fermentation conditions (70 h at 30 degrees C), the mass flux rose to 650 g h(-1) m(-2) using a disk equipped with 2 mm vanes rotating at 2000 rpm against 380 g h(-1) m(-2) with a smooth disk at the same speed. The maximum flux observed was 1560 g h(-1) m(-2) with a 6-mm vanes disk rotating at 3000 rpm and a 36 degrees C broth. An interesting finding was that the permeate flux J(f) for various disks can be correlated by the same function of the mean shear stress at the membrane tau(wm) according to J(f) = 4.6 tau(wm) (0.717) for a 30 degrees C broth, showing that the effect of vanes is merely to increase the shear stress by raising the fluid core velocity between the membrane and the disk. With 6-mm vanes the core angular velocity was found to be 84% of disk velocity vs. 45% for a smooth disk. When the fermentation temperature was increased to 36 degrees C to produce a lower molecular weight polymer, the permeate flux rose by about 250%, much more than what could be expected from the reduction in permeate viscosity and followed the same power law with membrane shear stress as for 30 degrees C. The same device was equipped with a PES 50 kDa membrane to concentrate EPS by ultrafiltration. Permeate fluxes were of the order of 160 L h(-1) m(-2) at 2000 rpm and 30 degrees C with nearly complete EPS rejection. Finally, the net electrical power consumed by the disk was measured by subtracting the power consumed without fluid from the power during filtration at the same speed. This power increases with speed and with the presence of vanes, but since the gain provided by the vanes is very high, the specific energy per m(3) of permeate is minimal with the highest vanes tested (6 mm) and maximal for smooth disks.     

Effects of by-products of fermentation of banana pseudostem on ethanol separation by pervaporation

In this work, we performed recovery of ethanol from a fermentation broth of banana pseudostem by pervaporation (PV) as a lower-energy-cost alternative to traditional separation processes such as distillation. As real fermentation systems generally contain by-products, it was investigated the effects of different components from the fermentation broth of banana pseudostem on PV performance for ethanol recovery through commercial flat sheet polydimethylsiloxane (PDMS) membrane. The experiments were compared to a binary solution (ethanol/water) to determine differences in the results due to the presence of fermentation by-products. A real fermented broth of banana pseudostem was also used as feed for the PV experiments. Seven by-products from fermented broth were identified: propanol, isobutanol, methanol, isoamyl alcohol, 1-pentanol, acetic acid, and succinic acid. Moreover, the residual sugar content of 3.02 g/L¹ was obtained. The presence of methanol showed the best results for total permeate flux (0.1626 kg·m⁻²·h⁻¹) and ethanol permeate flux (0.0391 kg·m⁻²·h⁻¹) during PV at 25°C and 3 wt% ethanol, also demonstrated by the selectivity and enrichment factor. The lowest total fluxes of permeate were observed in the experiments containing the acids. Better permeance of 0.1171 from 0.0796 kg·m⁻²·h⁻¹ and membrane selectivity of 9.77 from 9.30 were obtained with real fermentation broth than with synthetic solutions, possibly due to the presence of by-products in the multicomponent mixtures, which contributed to ethanol permeation. The results of this work indicate that by-products influence pervaporation of ethanol with hydrophobic flat sheet membrane produced from the fermented broth of banana pseudostem.     

Assessment of in-line near-infrared spectroscopy for continuous monitoring of fermentation processes

The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain.     

Fermentative production of dl-lactic acid from amylase-treated rice and wheat brans hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp.

Rice and wheat brans, without additional nutrients and hydrolyzed by alpha-amylase and amyloglucosidase, were fermented to DL-lactic acid using a newly isolated strain of Lactobacillus sp. RKY2. In batch fermentations at 36 degrees C and pH 6, the amount of lactic acid in fermentation broth reached 129 g l(-1) by supplementation of rice bran with whole rice flour. The maximum productivity was 3.1 g lactic acid l(-1) h(-1) in rice bran medium supplemented with whole rice flour or whole wheat flour.     

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.     

Propionic Acid Production by a Propionic Acid-Tolerant Strain of Propionibacterium acidipropionici in Batch and Semicontinuous Fermentation

A propionic acid-tolerant derivative of Propionibacterium acidipropionici P9 was obtained by serially transferring strain P9 through broth that contained increasing amounts of propionic acid. After 1 year of repeated transfers, a strain (designed P200910) capable of growth at higher propionic acid concentrations than P9 was obtained. An increase in the proportion of cellular straight-chain fatty acids and uncoupling of propionic acid production from growth were observed for strain P200910. Growth rate, sugar utilization, and acid production were monitored during batch and semicontinuous fermentations of semidefined medium and during batch fermentation of whey permeate for both strain P200910 and strain P9. The highest propionic acid concentration (47 g/liter) was produced by P200910 in a semicontinuous fermentation. Strain P200910 produced a higher ratio of propionic acid to acetic acid, utilized sugar more efficiently, and produced more propionic acid per gram of biomass than did its parent in all fermentations.     

Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3.4-22.5 g kg(-1) biomass) and the power-law model adequately described the rheological properties. In the cultivations there were pellets, clumps, and freely dispersed hyphal elements. The different morphological fractions were quantified using image analysis. The apparent viscosity of the fermentation broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations.     

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.     

Extractive acetonobutylic fermentation by coupling ultrafiltration and distillation

An extractive acetonobutylic fermentation process is developed by integrating bioproduction, Ultrafiltration, and distillation, providing simultaneous retention of biomass, selective removal of inhibitors from the permeate, as well as separation and purification of acetone-butanol-ethanol solvents. Successive batch fermentations were performed with normal pressure distillation (98 degrees C), which permitted prolonging and enhancing (by a factor of 3) solvent production, with very few volume exchanges of medium (average dilution rate ws 0.002 h(-1)), and recovering on-line concentrated solvents. Different operating conditions were also tested in order to study the presence of extracellular autolytic enzymes as inhibition factors: It was shown that, (1) extracellular autolytic activity remains low during the larger part of fermentations, even without enzyme-inactivating thermotreatment in the distillation boiler, and (2) high-temperature distillation causes deleterious effects to the culture medium for long duration treatments. Progressive improvements of the process were achieved, first, by managing continuous runs, providing a minimum renewal of the culture medium and, mainly, by decreasing temperature and pressure of distilation. Solvent productivity then reached 2.6 g/L h for a 0.036 h(-1) average dilution rate, corresponding to a feed concentration of 156 g/L glucose actually consumed.     

Purification during crossflow electromicrofiltration of fermentation broth

The present study was to investigate the purification of a fermentation broth by an electromicrofiltration membrane. Microfiltration runs with a crude and a centrifuged broth, with solution of particles recovered from centrifugation and with permeates from microfiltration experiments were thus compared.Microfiltration performances were governed by colloids and small particles that induced sharp initial flux declines. For these results, the evolution of the overall membrane resistance was increased by 80% in comparison with the electromicrofiltration membrane. The main focus of this study was set on the enhancement of the filtrate flux by an electric field. This pressure electrofiltration leads to a drastic improvement of the filtration by 100% and the filtration time was thereby reduced. Pressure electrofiltration serves as an interesting alternative to the cross-flow filtration and it effectively separates advantageous constituents such as amino acids and biopolymers from a fermentation broth. They were equally maintained during the microelectrofiltration, although they were significantly reduced by 45% by the microfiltration without the application of an electric field. Accordingly, since the electrofiltration membrane was provided more permeability, this study experimentally demonstrates that the permeability inside a membrane can be controlled using an electric field.     

Effect of biomass concentration and mycelial morphology on fermentation broth rheology

The effect of biomass concentration and mycelial morphology on fungal fermentation broth rheological properties has been investigated. In previous work it had been shown that commonly used rheological parameters, such as the power law consistency and flow behavior indices, could be correlated successfully with the broth biomass concentration and clump morphological parameters of roughness and compactness. More recent work on a broader range of data showed a correlation between roughness and compactness; consequently, it was not correct to use both of these morphological variables simultaneously in rheological parameter correlations. Furthermore, earlier correlations were only made using clump morphological parameters, as clumps were found to be around 90% of the biomass in batch fermentations. In the present work it was found that the percentage of clumps fell to around 30% to 40% of a sample during the later stages of fed-batch fermentations. No clear relationship between the flow behavior index and biomass concentration was found, at least for those phases of the fermentation in which the viscosities were high enough for the rheology to be characterized by a disk turbine rheometer. The mean value of the flow behavior index was found to be 0.35 +/- 0.1 (standard deviation) throughout both batch and fed-batch fermentations, although some significant deviations from this value were observed early and very late in the fermentations. Correlations for the consistency index, measured using a disk turbine rheometer, were based on the biomass concentration and the mycelial size (represented by the mean projected area or the mean maximum dimension of all the mycelia). These correlations were reasonably successful for both fed-batch and batch fermentations. The correlation using the mean maximum dimension was preferred to that using the mean projected area, as the former is independent of magnification. The proposed correlation is: where K is the consistency index (Pa. s(n>)), C(m) is the biomass concentration as dry cell weight (g L(-1)), and D is the mean maximum dimension (microm). It should be noted that small changes in the exponent on the biomass concentration (alpha) may dramatically affect any predictions. Consequently, caution in the use of this correlation (and that based on mean projected area) is advocated, although its accuracy may be suitable for operational or design purposes.     

Production of solvents (ABE fermentation) from whey permeate by continuous fermentation in a membrane bioreactor

A continuous bioreactor where cells were recycled using a cross-flow microfiltration (CFM) membrane plant was investigated for the production of solvents (ABE fermentation) from whey permeate using Clostridium acetobutylicum P262. A tubular CFM membrane plant capable of being backflushed was used.The continuous fermentations were characterized by cyclic solventogenic and acidogenic behaviour, and ultimately degenerated to an acidogenic state. Steady-state solvent production was obtained for only short periods. This degeneration is attributed to the complex morphological behaviour of this strain of organism on this substrate.It is postulated that to achieve steady-state solvent production over extended periods of time, it is necessary to maintain a balance among the various morphological cell forms, i.e. acid-producing vegetative cells, solvent-producing clostridial cells, and inert forms, e.g. spores.     

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.     

Broth conditions determining specific cake resistance during microfiltration of Bacillus subtilis

The effects of broth pH, pressure, temperature, and fermentation medium on specific cake resistance were studied for dead-end microfiltration of Bacillus subtilis. Decreases in pH and transmembrane pressure decreased the specific cake resistance for cells grown in both complex and defined media. With the complex medium, the reduction in resistance with temperature decrease did not offset the flux decrease caused by the increase in viscosity. The greatest decrease in specific cake resistance occurred with adjustment of pH to 7.5 for cells grown in defined medium. For those cells the change in pH resulted in aggregation leading to a large increase in flux.     

Submerged fermentation in wheat substrates for production of Monascus pigments

Cereal grains are normally used as solid substrates for the production of Monascus metabolites. However, solid fermentation in these substrates requires complex control systems, whereas in liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods. A flour composed of a mixed variety of Canadian hard wheat was used as sole nutrient source to produce the pigments of Monascus purpureus Went (IMI 210765). Supplementation with NH₄Cl promoted biomass and orange dye formation, whereas the use of zinc sulphate favoured red dyes production. In submerged fermentations significant differences in final pigment yields were observed in the use of wheat-based broth at different concentrations in the presence of bran particles and/or gluten protein. It has been found that the viscosity of the broth had a significant effect on the growth morphology and production of pigments. Gluten-free wheat flour at concentrations of 3–5% was found to be the most suitable for liquid Monascus culture. The subsequent use of passive immobilization of Monascus served to enhance red pigment yields and to facilitate the downstream processing of the dyes.     

Ethanol separation from molasses based fermentation broth by reverse osmosis

Summary Irradiated styrene-grafted cellulose acetate membrane was used for the separation of ethanol by reverse osmosis. Ethanol separation from molasses based fermentation broth resulted in separation efficiency of 90% at an operating pressure of 1400 psig. Lower permeate flux was observed with fermented broth compared to aqueous ethanol.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.