Extracellular enzyme loss during polyelectrolyte flocculation of cells from fermentation broth

Abstract

Association of extracellular protein product with flocculated cells reduces product yield. Here, partitioning of the enzyme subtilisin between the liquid and polyelectrolyte-flocculated and sedimented Bacillus increased as the polymer dosage was increased beyond that necessary to obtain optimum floc character (brain floc) for cell removal by centrifugation. Partitioning to the cell floc is partly physical entrapment at all polymer dosages; however, at higher levels there is also direct interaction between the polyelectrolyte and enzyme. Enzyme loss was not likely due to pH denaturation during the flocculation process because conditions were within the stable pH range of the enzyme. The direct interaction between polyelectrolyte and enzyme was characterized through turbidimetric titrations and partitioning studies. Neither changes in the polymer feed concentration nor the method of polymer addition reduced the enzyme loss at dosages optimal for cell removal.

Integrated ultra scale-down and multivariate analysis of flocculation and centrifugation for enhanced primary recovery

“…The reduction of impurities by flocculation and centrifugation can enhance further downstream processes (Balasundaram et al, 2009;Singh et al, 2016;Felo et al, 2015). However, Pearson et al, observed enzyme losses during polyelectrolyte flocculation and explained possible reasons for these losses (Pearson et al, 2004b). According to their study, the product can either interact with the flocculant or be entrapped in the flocs, leading to changes in product yield.…”

“…As already concluded from the sequential analysis, the feed variability influenced flocculation and centrifugation performance. Pearson et al, already identified batch variability as the most important factor for enzyme loss variability (Pearson et al, 2004b). Barany & Szepesszentgyörgyi detected an influence of suspension concentration and composition on flocculation efficiency, showing that suspension characteristics and content may vary from batch-to-batch (Barany and Szepesszentgyörgyi, 2004).…”

“…The influence of shear stress and feed variability on flocculation and centrifugation is well known (Chatel et al, 2014;Barany and Szepesszentgyörgyi, 2004;Pearson et al, 2004b). This work's novelty is the application of PCA and PLS to study the multi-parameters effect on flocculation and centrifugation.…”

Improving permeate flux and product transmission in the microfiltration of a bacterial cell suspension by flocculation with cationic polyelectrolytes

“…Batch to batch variability in the fermentation broth was controlled by using broth from the same fermentation for all of the experiments where direct comparisons were made; however, several batches of fermentation broth were used with similar results obtained for different broths at the same experimental conditions. The engineered subtilisin has a molecular weight of 28 kDa and a pI of 6.8 giving it a positive charge at the filtration pH of 6.5 [38]. Table 1 gives the characteristics of the cationic polyelectrolytes used for these experiments (Kemira Water Solutions, Lakeland, FL).…”

Bioprocess Engineering Issues That Would Be Faced in Producing a DNA Vaccine at up to 100 m3 Fermentation Scale for an Influenza Pandemic

“…Classical filtration with a bacterium such as E. coli is constrained by cell size. Flocculating agents have been applied but tend to interfere with the product (52). Some lysis of E. coli harvested by disk centrifuges has been observed (53, 54).…”