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J Ind Microbiol Biotechnol (2014) 41:647655 DOI 10.1007/s10295-014-1405-8
BIOTECHNOLOGY METHODS
In situ pH management for microbial culture in shake asks and its application to increase plasmid yield
Reenu Sanil Vishwanathgouda Maralingannavar
Mugdha Gadgil
Received: 2 September 2013 / Accepted: 10 January 2014 / Published online: 30 January 2014 Society for Industrial Microbiology and Biotechnology 2014
Abstract Shake asks are widely used to culture micro-organisms, but they do not allow for pH control without additional infrastructure. In the presence of a carbon source like glucose, culture pH typically decreases due to overow metabolism and can limit the growth of microorganisms in shake asks. In this study, we demonstrate the use of magnesium hydroxide-loaded pH managing hydrogels (mpHmH) for in situ base release to counter the decrease in culture pH in shake asks using Escherichia coli as a model organism, in both complex and mineral salts medium. Base release from m-pHmH is shown to increase with decreasing pH (22-fold increase in release rate from pH 8 to 5), thus providing feedback from culture pH. The addition of m-pHmH resulted in better pH maintenance and higher biomass yields of E. coli K12 in media containing glucose as a carbon source. The use of m-pHmH with additional buffer resulted in pH being maintained above 6.9 while pH decreases below 5 without m-pHmH. We demonstrate one application of such in situ pH management to increase the volumetric plasmid yield from E. coli in shake ask culture. In situ glucose release through a hydrogel to mimic fed-batch culture along with the addition of m-pHmH resulted in a 395 % increase in volumetric plasmid yield to 38 lg/ml in shake ask culture.
Keywords In situ pH control Shake ask culture
pH-responsive base release Magnesium hydroxide
Introduction
Microbial cell growth is affected by pH, which can decrease signicantly during culture in the presence of carbon sources like glucose due to overow metabolism [19]. A change in pH beyond the permissive range of the organism affects cell growth. For example, the growth of Escherichia coli M23 was shown to cease below a pH of3.9 [11]. It is estimated that more than 90 % of all cultures in biotechnology are carried out in shake asks [1], which traditionally have no control over pH. A decrease...