Application of hydrocyclone for separation of Pichia pastoris produced r-HBsAg from fermentation culture: impact of concentration and pressure on hydrocyclone performance

Abstract

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated.

Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R² = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3 bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19 mm and 21 mm were used for separating the yeast cells.

Keywords:

• Concentration ratio
• hydrocyclone
• Pichia pastoris
• separation
• three level factorial design
• underflow
• yeast

Acknowledgments

We are particularly grateful to Dr. Maryam Khatami and Dr. Parisa Naeiji for their constructive feedback and comments.

Disclosure statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Nomenclature

ANOVA	=	Analysis of variance
r-HBsAg	=	Recombinant of Hepatitis B surface antigen
C.V.	=	Coefficient of variation
C	=	Feed concentration
Cu	=	Under flow concentration
Rc	=	Solid concentration ratio
V	=	Feed inlet speed
R	=	Radius of cylindrical region
G	=	Force of gravity
LPM	=	Liter per minute
TLFD	=	Three level factorial design
PVC	=	Polyvinyl chloride
Inc	=	Incorporation
DOE	=	Design of experiments
DOF	=	Degree of freedom
SOS	=	Sum of square
MOS	=	Mean of square