Continuous foaming for protein recovery: part II. Selective recovery of proteins from binary mixtures

Foam separation may have potential for protein recovery. However, for foam separation to be a viable protein recovery technique it is important to demonstrate, not only that high enrichments and recoveries can be achieved for single proteins, but also that high enrichments and recoveries, together with selectivity of partition, can be achieved for recovery from multi-component mixtures. Most process streams which require purification are indeed complex multi-component mixtures, for example, fermentation broths. In this study, three binary protein mixtures were chosen for continuous foam separation: beta-casein:lysozyme; Bovine serum albumin (BSA):lysozyme and beta-casein:BSA (mixtures 1, 2, and 3, respectively). For each of these mixtures, the expected outcome of each experiment, based on a previous knowledge and determination of relevant protein physical properties, was that the first protein should be preferentially separated into the foam phase. On the basis of results reported in Part I of this study for the continuous foam separation of beta-casein, conditions found to favor maximum enrichment were selected. For each mixture a range of concentrations of both proteins was considered. For mixture 1, maximum protein recoveries in the foam phase were 85.6% and 25% for beta-casein and lysozyme, respectively; and for mixture 2, maximum recoveries of 77. 6% and 18.9% were obtained for BSA and lysozyme, respectively. Maximum enrichment ratios in the foam phase were 79.4 and 2.5 for beta-casein and lysozyme respectively in mixture 1; and 74.0 and 1.4 for BSA and lysozyme respectively in mixture 2. Selective partitioning of beta-casein and BSA into the foam phase was obtained in mixtures 1 and 2, respectively, particularly for protein concentrations at which dilute protein films are known to form at the gas-liquid interface in the foam. Maximum partition ratios for mixtures 1 and 2 were 31.8 and 52.8, respectively. For mixture 3, both BSA and beta-casein were enriched into the foam phase. Maximum enrichments were 42.9 and 24.7 for BSA and beta-casein, respectively; however, selective partitioning in mixture 3 was limited (maximum partition ratio being 1.8).     

Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams

Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc.     

Foam fractionation of globular proteins

Foam fractionation of bovine serum albumin (BSA) was studied as a model system for potato wastewater. The effects of feed concentration, superficial gas velocity, feed flow rate, bubble size, pH, and ionic strength on the enrichment and recovery of BSA were investigated in a single-stage continuous foam fractionation column. Enrichments ranged from 1.5 to 6.0 and recoveries from 5 to 85%. The feed concentrations were varied from 0.01 to 0.2 wt %, and enrichments were found to increase with lower feed concentrations. Enrichments also increased with lower superficial gas velocities and larger bubble sizes. At sufficiently low feed flow rates, enrichment was found to increase with an increase in the flow rate, eventually becoming insensitive to the feed flow rate at higher values. The pH was varied from 3.5 to 7.0 and ionic strength from 0.001M to 0.2M. The effects of pH and ionic strength were found to be coupled with bubble size. A minimum bubble size was found at pH 4.8, the isoelectric point of BSA, resulting in a minimum in the enrichment. Bubble size, and thus enrichment, was found to increase as the ionic strength decreased from 0.2M to 0.01M. Previous models(1,2) for the hydrodynamics of foam column were extended for a singlestage continuous foam fractionation column for the prediction of enrichment and recovery. The model assumed adsorption equilibrium, infinite surface viscosity, and bubbles of the same size. Though coalescence was formally accounted for in the model by considering bubble size as a function of foam height, calculations for the experimental runs were performed only for the case of no coalescence. Quantitative predictions of enrichment and recovery could not be made with a single representative bubble size because of the broad inlet bubble size distribution as well as broadening of the distribution as a result of coalescence. The experimental enrichments were higher and recoveries were lower than the model predictions, the discrepancy being more pronounced at lower feed concentrations because of increased coalescence. The higher enrichments are due to the predominant effect of internal reflux as a result of coalescence whereas the lower recoveries are a result of detrimental effects of broadening bubble size distributions.     

Effect of invertase on the batch foam fractionation of bromelain

Foam fractionation can be used to enrich a hydrophobic protein such as bromelain from an aerated dilute protein solution because the protein foams. On the other hand, a protein such as invertase, which is hydrophilic, is not likely to foam under similar aerated conditions. While a foam fractionation process may not be approapriate for recovering a hydrophilic protein alone, it is of interest to see how that non-foaming protein affects the foaming protein when the two are together in a mixture. The bromelain enrichment, activity and mass recovery were observed as a function of the solution pH in order to explore how invertase can affect the recovery of bromelain in a foam fractionation process.     

Anaerobic digestion foaming causes – A review

Anaerobic digestion foaming has been encountered in several sewage treatment plants in the UK. Foaming has raised major concerns for the water companies due to significant impacts on process efficiency and operational costs. Several foaming causes have been identified over the past few years by researchers. However, the supporting experimental information is limited and in some cases absent. The present report aims to provide a detailed review of the current anaerobic digestion foaming problem and to identify gaps in knowledge regarding the theory of foam formation in anaerobic digesters.     

Quantitative use of fluorescent in situ hybridization to examine relationships between mycolic acid-containing actinomycetes and foaming in activated sludge plants

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P < 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m(3) completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 x 10(6) cells ml(-1) or 4 x 10(12) cells m(-2). We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.     

The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

The effect of Bacillus subtilis producing Surfactin in ROS production and transformation efficiency of tobacco cells

Surfactin secreted by bacilli has biological functions in plant. Surfactin C14 and C15 have the highest effect on inducing hydrogen peroxide species release in the plant. Surfactin production in the two Bacillus strains ACCT21332 and FKR3 were analysed by HPLC and the phytotoxicity of the Bacilli-derived surfactins was determined in Tobacco cell culture. Surfactin C14 and C15 were detected in ACCT21332 but not in FKR3 strain. Extracellular hydrogen peroxide produced by tobacco cell culture cells exposed to ATCC21332 and FKR3 strains increased compared to untreated ones. The Agrobacterium mediated transformation rate of tobacco cells drops from 4% transformed cells to 0.8 and 1.2% when pretreated with ATCC21332 or FKR3 strain, respectively. The strong drop in transformation rate of plant cell culture after FKR3 strain pre-treatment indicates that Surfactin C14 and C15 are not the major or the only cause in protecting plant cells from Agrobacterial infection and transformation.     

The effect of organic loading rate on foam initiation during mesophilic anaerobic digestion of municipal wastewater sludge

The impact of increasing organic load on anaerobic digestion foaming was studied at both full and bench scale. Organic loadings of 1.25, 2.5 and 5 kg VS m⁻³ were applied to bench-scale digesters. Foaming was monitored at a full scale digester operated in a comparable organic loading range over 15 months. The bench scale batch studies identified 2.5 kg VS m⁻³ as a critical threshold for foam initiation while 5 kg VS m⁻³ resulted in persistent foaming. Investigation of a full scale foaming event corroborated the laboratory observation that foaming may be initiated at a loading rate of ?2.5 kg VS m⁻³. Experimental findings on foam composition and differences in the quality characteristics between foaming and non-foaming sludges indicated that foam initiation derived from the combined effect of the liquid and gas phases inside a digester and that the solids/biomass ultimately stabilized foaming.     

Evaluation of different Bacillus strains in respect of their ability to produce Surfactin in a model fermentation process with integrated foam fractionation

Biosurfactants increasingly gain attention due to the manifold of possible applications and production on the basis of renewable resources. Owing to its various characteristics, Surfactin is one of the most studied biosurfactants. Since its discovery, several Surfactin producers have been identified, but their capacity to produce Surfactin has not been evaluated in a comparison. Six different Bacillus strains were analyzed regarding their ability to produce Surfactin in model fermentations with integrated foam fractionation, for in situ product enrichment and removal. Three of the investigated strains are commonly used in Surfactin production (ATCC 21332, DSM 3256, DSM 3258), whereas two Bacillus strains are described for the first time (DSM 1090, LM43a50°C) as Surfactin producers. Additionally, the Bacillus subtilis type strain DSM 10^T was included in the evaluation. Interestingly, all strains, except DSM 3256, featured high values for Surfactin recovered from foam in comparison to other studies, ranging between 0.4 and 1.05 g. The fermentation process was characterized by calculating procedural parameters like substrate yield Y _X/S, product yield Y _P/X, specific growth rate μ, specific productivity q _Surfactin, volumetric productivity q _Surfactin, Surfactin and bacterial enrichment as well as Surfactin recovery. The strains differ most in specific and volumetric productivity; nevertheless, it is evident that it is not possible to name a Bacillus strain that is the most appropriate for the production of Surfactin under these conditions. In contrast, it becomes apparent that the choice of a specific strain should depend on the applied fermentation conditions.     

Protein separation by differential drainage from foam

Commercially available beer, which is a dilute solution containing components of yeast, malt, and hop used in the manufacture of the beer, was used as a model system to demonstrate the potential of foam fractionation beyond the primary foaming stage. Most of the components present in the beer concentrated in the initial foam, but they drained differentially in the subsequent collapsed foam collected over a period of 30 min. This resulted in further enrichment, in particular, of components which were present in low concentration in the original beer, Preferential drainage from foam, hence, might provide a novel way of fractionating further the proteins concentrated initially in the liquid films of foam. (c) 1994 John Wiley & Sons, Inc.     

发酵产泡性能降低的解脂耶氏酵母菌株的获得及其评价

微生物发酵过程中泡沫的产生是发酵领域遇到的共性问题。在不影响发酵性能的前提下抑制菌株的产泡,对简化操作以及降低发酵成本具有较为重要的意义。解脂耶氏酵母(Yarrowia lipolytica,之前称为Candida lipolytica)是一种常用的合成生物学底盘,也是合成赤藓糖醇等功能糖醇的生产菌株。但在发酵合成赤藓糖醇的过程中会产生大量的泡沫,需要添加消泡剂以消除泡沫。【目的】本研究旨在开发一种产泡能力显著降低的解脂耶氏酵母新菌株,以减少赤藓糖醇发酵过程中消泡剂的添加。【方法】本研究利用解脂耶氏酵母中非同源靶向重组占支配地位的原理,采用一段外源DNA随机插入基因组的手段,随机突变基因组,改变菌株的发酵产泡性能,使突变株在发酵过程中不产泡或者降低其产泡的能力。【结果】通过筛选,获得一株在发酵过程中产泡性能显著降低的工程菌株,该菌株在保留高效合成赤藓糖醇性能的同时,显著降低了泡沫的产生。【结论】所获得的菌株对工业发酵合成赤藓糖醇具有较为重要的意义,也为控制其他微生物发酵过程中泡沫的生成提供了思路。     

OBSERVATIONS ON FOAMING AND ITS INHIBITION IN A BACTERIAL CULTURE

SUMMARY: Inhibition of foaming in a continuous culture of bacteria has been studied. At first, foaming was inhibited by the addition of antifoam to the culture only when a foam layer was present. When the antifoam was added in this way foaming often became intense and antifoam additions had to be more and more frequent. In a preferred method the antifoam was added at regular intervals which were sufficiently short to inhibit foaming completely throughout the intervening periods. This method required less antifoam than the other. The effects of foaming in cultures, its causes, and allied problems are discussed.     

Quantitative Use of Fluorescent In Situ Hybridization To Examine Relationships between Mycolic Acid-Containing Actinomycetes and Foaming in Activated Sludge Plants

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P << 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m³ completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 × 10⁶ cells ml⁻¹ or 4 × 10¹² cells m⁻². We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.     

Characterisation of HFBII biosurfactant production and foam fractionation with and without antifoaming agents

The effects of foaming on the production of the hydrophobin protein HFBII by fermentation have been investigated at two different scales. The foaming behaviour was characterised in standard terms of the product enrichment and recovery achieved. Additional specific attention was given to the rate at which foam, product and biomass overflowed from the fermentation system in order to assess the utility of foam fractionation for HFBII recovery. HFBII was expressed as an extracellular product during fed-batch fermentations with a genetically modified strain of Saccharomyces cerevisiae, which were carried out with and without the antifoam Struktol J647. In the presence of antifoam, HFBII production is shown to be largely unaffected by process scale, with similar yields of HFBII on dry matter obtained. More variation in HFBII yield was observed between fermentations without antifoam. In fermentations without antifoam, a maximum HFBII enrichment in the foam phase of 94.7 was measured with an overall enrichment, averaged over all overflowed material throughout the whole fermentation, of 54.6 at a recovery of 98.1%, leaving a residual HFBII concentration of 5.3 mg L⁻¹ in the fermenter. It is also shown that uncontrolled foaming resulted in reduced concentration of biomass in the fermenter vessel, affecting total production. This study illustrates the potential of foam fractionation for efficient recovery of HFBII through simultaneous high enrichment and recovery which are greater than those reported for similar systems.     

Effect of pH on successive foam and sonic droplet fractionation of a bromelain-invertase mixture

A droplet fractionation method was previously developed to concentrate a dilute nonfoaming protein solution. In that earlier study with invertase, it was demonstrated that droplets created by ultrasonic energy waves could be enriched up to 8 times that of the initial dilute invertase solution. In this study, a mixture of bromelain (a foaming protein) and invertase (a nonfoaming protein) is investigated as a preliminary step to determine if droplet fractionation can also be used to separate a non-foaming protein from foaming proteins. The foaming mixture containing bromelain is first removed by bubbling the binary mixture with air. After the foam is removed, the protein rich air-water interfacial layer is skimmed off (prior to droplet fractionation) so as not to interfere with the subsequent droplet production from the remaining bulk liquid, rich in non-foaming protein. Finally, sonic energy waves are then applied to this residual bulk liquid to recover droplets containing the non-foaming protein, presumed to be invertase. The primary control variable used in this droplet fractionation process is the pH, which ranged for separate experiments between 2 and 9. It was observed that the maximum overall protein partition coefficients of 5 and 4 were achieved at pH 2 and 4, respectively, for the initial foaming experiment followed by the post foaming droplet fractionation experiment.     

The mechanism of stabilization of actinomycete foams and the prevention of foaming under laboratory conditions

Summary Cultures ofNocardia amarae give rise to cell-stabilized foams in a laboratory scale foaming apparatus. The organism produces a surfactant and the cells are very hydrophobic; factors which, in terms of froth flotation theory, are essential for foam production and transport of the cells from the aqueous to the bubble phase. The addition of montmorillonitic clay to the culture prior to foaming prevents foam stabilization. The results obtained suggest the formation of a salt-dependent, reversible, bacterium-montmorillonite complex which prevents transport of cells to the bubble phase.     

Foaming of proteins: New prospects for enzyme purification processes

Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the “White Biotechnologies” for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique.     

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.     

Direct inoculation into media containing bile salts and antibiotics is unsuitable for the detection of acid/salt stressed Escherichia coli O157:H7

The efficiency of selective enrichment broths for the recovery of low numbers of acid/salt stressed Escherichia coli O157:H7 was determined. Stressed cultures were diluted to low levels and recovered in tryptone soya broth with added bile salts, to make modified tryptone soya broth, and buffered peptone water with various combinations of antibiotic supplementation including novobiocin, acriflavine and a mixture of vancomycin, cefsulodin and cefixime (VCC) at 37 °C and 42 °C. Significantly fewer stressed cells, in some cases as little as 0·3% of the starting population, were recovered by all the selective enrichment broths containing bile salts or VCC antibiotics compared to the non-selective controls. The use of such enrichments to recover low numbers of stressed E. coli O157:H7 may result in failure to detect the organism. Parallels with salmonella methodology are made and the need for a non-selective pre-enrichment stage in E. coli O157:H7 methods discussed.