Ultra scale‐down characterization of the impact of conditioning methods for harvested cell broths on clarification by continuous centrifugation—Recovery of domain antibodies from rec E. coli

The processing of harvested E. coli cell broths is examined where the expressed protein product has been released into the extracellular space. Pre‐treatment methods such as freeze–thaw, flocculation, and homogenization are studied. The resultant suspensions are characterized in terms of the particle size distribution, sensitivity to shear stress, rheology and solids volume fraction, and, using ultra scale‐down methods, the predicted ability to clarify the material using industrial scale continuous flow centrifugation. A key finding was the potential of flocculation methods both to aid the recovery of the particles and to cause the selective precipitation of soluble contaminants. While the flocculated material is severely affected by process shear stress, the impact on the very fine end of the size distribution is relatively minor and hence the predicted performance was only diminished to a small extent, for example, from 99.9% to 99.7% clarification compared with 95% for autolysate and 65% for homogenate at equivalent centrifugation conditions. The lumped properties as represented by ultra scale‐down centrifugation results were correlated with the basic properties affecting sedimentation including particle size distribution, suspension viscosity, and solids volume fraction. Grade efficiency relationships were used to allow for the particle and flow dynamics affecting capture in the centrifuge. The size distribution below a critical diameter dependant on the broth pre‐treatment type was shown to be the main determining factor affecting the clarification achieved. Biotechnol. Bioeng. 2014;111: 913–924. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Flocculation in brewing yeasts: a computer simulation study

A new simulator, INDISIM-FLOC, based on the individual-based simulator INDISIM, is used to examine the predictions of two different models of yeast flocculation. The first, proposed by Calleja is known as the "addition" model. The second, proposed by Stratford is known as the "cascade" model. The simulations show that the latter exhibits a better qualitative agreement with available experimental data.     

The use of succinic acid, as a pH buffer, expands the potentialities of utilisation of a chemically defined medium in Saccharomyces cerevisiae flocculation studies

The pH, viability and flocculation during the growth of Saccharomyces cerevisiae NCYC 1214 in rich medium (YEPD) and in chemically defined medium (YNB) were compared. As the pH fell from 5.3 to 2.2, the viability fell to 20% and no flocculation was observed in YNB medium, despite the high flocculation in buffer. With the addition of 50 mM succinic acid and 3.1 mM Ca²⁺, similar growth to YEPD, with 99% of viability and flocculation in culture medium, was obtained.     

Harvesting of Arthrospira platensis by flocculation with phytic acid from rice bran

ABSTRACT

The recovery of algal biomass is one of the critical steps involved in the commercial production of beneficial metabolites from Arthrospira platensis. Efficient and safe harvesting methods that do not sacrifice quality of final product are important for commercial application. Phytic acid (PA) is a natural non-toxic phytochemical widely distributed in plant tissues. Effect of PA from rice bran on the growth, trichome morphology such as spiral number and algal filament length, and harvesting efficiency of A. platensis were investigated. Cells aggregated into large cell flocs after the addition of PA in the medium, and algal spiral number and filament length increased. UV-vis spectra indicated the interactions between PA and algal cells. Adding PA at stationary growth phase is a good strategy for harvesting, since no adverse effect to biomass growth and harvesting efficiency. Harvesting efficiency of 95.69% at 0.5% (v/v) PA was superior to other conventional harvesting methodologies.     

Pretreatments for enhancing clarification efficiency of depth filtration during production of monoclonal antibody therapeutics

High cell density, high product titer mammalian cell culture is the new paradigm for production of recombinant proteins. While the typical motivation is to get a high product titer, additional undesirable outcomes often include an increase in percentage solids in the cell culture fluid (cellular debris and sub-micron colloids), thereby offering new challenges to downstream processing. This article focuses on scouting and comparison of different approaches used for clarification of cell culture fluid. The approaches include centrifugation followed by depth filtration, direct depth filtration without centrifugation and feed pretreatment with use of specially designed density gradient filtration to improve efficiency of clarification and removal of process contaminants from feed stream. The work also evaluates impact of three different pretreatment approaches, namely pH adjustment to acidic condition, metal cation (calcium phosphate) flocculation, and polycationic polymer flocculation (using polymer-I and polymer-II). The results obtained indicate that the use of pretreatment significantly improves the clarification efficiency of depth filtration. Pretreatment options like polycationic polymer-I based flocculation resulted in a >5 fold reduction in filter area requirement as well as >6 fold reduction in HCDNA while retaining acceptable recovery of the IgG (>98%). Thus, pretreatment offers a significant reduction in the depth filtration footprint (~5–6 fold decrease in filter area requirement). However, one must take into consideration the process development time required, capital cost, consumable cost, cost of the pretreatment chemical, cost of testing to demonstrate clearance of treatment agent, ease of scale-ability, and process robustness when finalizing the optimal clarification approach.     

On-line determination of flocculating Saccharomyces cerevisiae concentration and growth rate using a capacitance probe

During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method.     

Applications of yeast flocculation in biotechnological processes

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects—the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer's yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculentSaccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.     

Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis

Enzymes and mechanics play major roles in lignocellulosic biomass deconstruction in biorefineries by catalyzing chemical cleavage or inducing physical breakdown of biomass, respectively. At industrially relevant substrate concentrations mechanical agitation is also a driving force for mass transfer as well as agglomeration of elongated biomass particles. Contrary to the physically induced particle attrition, which typically facilitates feedstock handling, particle agglomeration tends to hinder mass transfer and in the worst case induces processing difficulties like pipe blockage. Understanding the complex interplay between mechanical agitation and enzymatic degradation during hydrolysis is therefore critical and was the aim of this study. Particle size analyses revealed that neither mechanical agitation alone nor enzymatic treatment without mechanical agitation had any noteworthy effect on flax fiber attrition. Similarly, successive treatment, where mechanical agitation was either preceded or proceeded by enzymatic hydrolysis, did not induce any substantial segmentation of flax fibers. Simultaneous enzymatic and mechanical treatment on the other hand was found to promote fast fiber shortening. Higher hydrolysis yields, however, were obtained from nonagitated samples after prolonged enzymatic treatment, indicating that mechanical agitation in the long run reduces activity of the cellulolytic enzymes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2754, 2019.