Characterization of respiratory physiology in Lactococcus lactis for high-yield production of high-performance starter cultures
Doctoral thesis, 2020
Fermented food products are consumed world-wide, every day, and the demand is growing as intake increases. Lactococcus lactis is a lactic acid bacterium commonly used as a starter culture to produce fermented dairy products. The quality of starter cultures is linked to the conditions during its production process and affects the culture activity during its use in dairy product fermentation. Therefore, industrial manufacturers of starter cultures strive to not only optimize biomass yield, but also the cell robustness. L. lactis starter cultures are produced by anaerobic fermentation or by aerobic respiration when haemin is available in the cultivation medium.
The aim of this work was to investigate the response of L. lactis to respiration-permissive conditions, to enable redesign and optimization of the batch production process. The process parameters were carefully chosen to mimic the industrial process. The effects of the specific growth rate on respiratory metabolism, energetics and cell quality were quantified using chemostat cultivations. Compared to anaerobic metabolism, the respiratory metabolism of L. lactis was remarkably flexible, and could be modulated by controlling the dilution rate. The lowest dilution rate supported full respiratory metabolism, whereas the higher dilution rates caused a shift towards respiro-fermentative metabolism. The inoculation procedures were investigated in detail to gain an understanding of the occurrence of lag phases after inoculation. It was found that the length of the lag phase in subsequent main cultures was related to galactose excretion in lactose-grown pre-cultures. Furthermore, based on lacS gene expression measurements in lactose-grown cultures, it is suggested that LacS is responsible for the galactose excretion as a galactose-lactose antiporter.
The quality of frozen and freeze-dried products was investigated and sensitivity to freeze-drying was found to be associated with the physiological state of the cells during cultivation. Cells harvested under respiration-permissive conditions in batch and chemostat cultivations at low dilution rate were less robust to freeze-drying, whereas higher dilution rates led to robust cells performing equally well or better than anaerobic cells.
The findings of this work underline the importance of systematically studying the combination of upstream and downstream aspects of production processes. The results indicate that by controlling the specific growth rate and the haemin concentration during the aerobic growth of L. lactis, not only higher biomass yields, but also better cell robustness, can be achieved.
The aim of this work was to investigate the response of L. lactis to respiration-permissive conditions, to enable redesign and optimization of the batch production process. The process parameters were carefully chosen to mimic the industrial process. The effects of the specific growth rate on respiratory metabolism, energetics and cell quality were quantified using chemostat cultivations. Compared to anaerobic metabolism, the respiratory metabolism of L. lactis was remarkably flexible, and could be modulated by controlling the dilution rate. The lowest dilution rate supported full respiratory metabolism, whereas the higher dilution rates caused a shift towards respiro-fermentative metabolism. The inoculation procedures were investigated in detail to gain an understanding of the occurrence of lag phases after inoculation. It was found that the length of the lag phase in subsequent main cultures was related to galactose excretion in lactose-grown pre-cultures. Furthermore, based on lacS gene expression measurements in lactose-grown cultures, it is suggested that LacS is responsible for the galactose excretion as a galactose-lactose antiporter.
The quality of frozen and freeze-dried products was investigated and sensitivity to freeze-drying was found to be associated with the physiological state of the cells during cultivation. Cells harvested under respiration-permissive conditions in batch and chemostat cultivations at low dilution rate were less robust to freeze-drying, whereas higher dilution rates led to robust cells performing equally well or better than anaerobic cells.
The findings of this work underline the importance of systematically studying the combination of upstream and downstream aspects of production processes. The results indicate that by controlling the specific growth rate and the haemin concentration during the aerobic growth of L. lactis, not only higher biomass yields, but also better cell robustness, can be achieved.
batch cultivations
lag phase
haemin/haem
respiration-permissive conditions
aerobic growth
Lactic acid bacteria
acidification activity
continuous cultivations
starter culture production
Lecture hall HA1, Hörsalsvägen 4, Chemistry building and via Zoom (Chalmers, Johanneberg campus). Please contact Gunilla Bankel gunilla.bankel@chalmers.se to obtain password for online participation.
Opponent: Professor Bas Teusink, System Biology Lab, AIMMS,Vrije Universitet Amsterdam, The Netherlands
Author
Anna Johanson
Chalmers, Biology and Biological Engineering, Industrial Biotechnology
Johanson, A., Olsson, L., Franzén, C. J. & Goel, A. Respiratory growth of Lactococcus lactis depends on hemin concentration and leads to impaired performance after freeze-drying
Johanson, A., Kovács J., Olsson, L., Goel, A., & Franzén, C. J. Hemin-activated respiration in Lactococcus lactis for high-yield biomass production depends on hemin concentrations
Respiratory Physiology of Lactococcus lactis in Chemostat Cultures and Its Effect on Cellular Robustness in Frozen and Freeze-Dried Starter Cultures
Applied and Environmental Microbiology,;Vol. 86(2020)
Journal article
Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures
Journal of Industrial Microbiology and Biotechnology,;Vol. 46(2019)p. 33-43
Journal article
Starter cultures are used in the making of the vast majority of cheese worldwide. The term ‘starter culture’ is used to describe the bacteria used to ‘start’ the transformation of milk into cheese. The use of starter cultures is beneficial in cheese-making for several reasons. Firstly, the lactic acid they produce acidifies the milk by turning its natural lactose, which is a sugar, into lactic acid. This causes the milk to curdle and split, helping to form the curd, which is the essential ingredient in cheese. Secondly, the starter cultures influence the texture, aroma and flavour of the cheese. Thirdly, the lactose and many other freely available nutrients in the milk are consumed by the growing lactic acid bacteria, making it difficult for pathogenic and spoilage bacteria to grow, besides being inhibited by the acid produced and the resulting low pH. Preventing the growth of ‘bad’ bacteria helps preserve the cheese and is one of the reasons why cheese keeps much longer than milk.
The bacteria used in starter cultures are often cultured in batch cultivation, after which the cells are separated and preserved, either by freezing or freeze-drying, before being sold to cheesemakers. The bacterial cells must remain viable and maintain a high acidification activity until they are added to the milk, which is why cellular robustness is important in achieving high performance starter cultures.
This research was carried out on the bacterium Lactococcus lactis, which is known for its high acidification performance. L. lactis has previously been regarded as a fermentative anaerobic microorganism, growing without oxygen, and producing mainly lactic acid. However, in the presence of oxygen and exogenous heme respiratory metabolism can be established, and the product profile is changed to acetate, acetoin and diacetyl, in addition to lactic acid. Respiration increases the biomass yield, leading to a high yield process.
The aim of this work was to investigate how cultivation conditions affect the physiology and robustness of L. lactis cells. Thereby gaining a better understanding, allowing the starter culture production process to be redesigned and optimized in such a way as to enable high-yield production of robust, high-performance starter cultures.
The bacteria used in starter cultures are often cultured in batch cultivation, after which the cells are separated and preserved, either by freezing or freeze-drying, before being sold to cheesemakers. The bacterial cells must remain viable and maintain a high acidification activity until they are added to the milk, which is why cellular robustness is important in achieving high performance starter cultures.
This research was carried out on the bacterium Lactococcus lactis, which is known for its high acidification performance. L. lactis has previously been regarded as a fermentative anaerobic microorganism, growing without oxygen, and producing mainly lactic acid. However, in the presence of oxygen and exogenous heme respiratory metabolism can be established, and the product profile is changed to acetate, acetoin and diacetyl, in addition to lactic acid. Respiration increases the biomass yield, leading to a high yield process.
The aim of this work was to investigate how cultivation conditions affect the physiology and robustness of L. lactis cells. Thereby gaining a better understanding, allowing the starter culture production process to be redesigned and optimized in such a way as to enable high-yield production of robust, high-performance starter cultures.
Subject Categories
Industrial Biotechnology
ISBN
978-91-7905-264-5
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4731
Publisher
Chalmers
Lecture hall HA1, Hörsalsvägen 4, Chemistry building and via Zoom (Chalmers, Johanneberg campus). Please contact Gunilla Bankel gunilla.bankel@chalmers.se to obtain password for online participation.
Opponent: Professor Bas Teusink, System Biology Lab, AIMMS,Vrije Universitet Amsterdam, The Netherlands