scholarly journals Respiratory Physiology of Lactococcus lactis in Chemostat Cultures and Its Effect on Cellular Robustness in Frozen and Freeze-Dried Starter Cultures

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Anna Johanson ◽  
Anisha Goel ◽  
Lisbeth Olsson ◽  
Carl Johan Franzén
Keyword(s):  
Lactococcus Lactis ◽  
Specific Growth ◽  
Starter Cultures ◽  
Respiratory Metabolism ◽  
Cultivation Conditions ◽  
Content Type ◽  
Chemostat Cultures ◽  
Freeze Dried ◽  
Specific Growth Rates

ABSTRACT In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D = 0.1 h−1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rates to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate, and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10% of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D = 0.1 h−1 were less robust after freeze-drying and had lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells. IMPORTANCE Lactococcus lactis is used in large quantities by the food and biotechnology industries. L. lactis can use oxygen for respiration if heme is supplied in the growth medium. This has been extensively studied in batch cultures using various mutants, but quantitative studies of how the cell growth affects respiratory metabolism, energetics, and cell quality are surprisingly scarce. Our results demonstrate that the respiratory metabolism of L. lactis is remarkably flexible and can be modulated by controlling the specific growth rate. We also link the physiological state of cells during cultivation to the quality of frozen or freeze-dried cells, which is relevant to the industry that may lack understanding of such relationships. This study extends our knowledge of respiratory metabolism in L. lactis and its impact on frozen and freeze-dried starter culture products, and it illustrates the influence of cultivation conditions and microbial physiology on the quality of starter cultures.

scholarly journals Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates

2016 ◽  
Vol 82 (15) ◽  
pp. 4570-4583 ◽  
Author(s):  
Corinna Rebnegger ◽  
Tim Vos ◽  
Alexandra B. Graf ◽  
Minoska Valli ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Growth Rates ◽  
Specific Growth ◽  
Energy Requirement ◽  
Maintenance Energy ◽  
Content Type ◽  
Chemostat Cultures ◽  
Zero Growth ◽  
Maintenance Energy Requirement ◽  
Specific Growth Rates

ABSTRACTThe yeastPichia pastorisis a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented forP. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value ofmSat near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than themSestimated from faster-growing chemostat cultures. This difference indicated thatP. pastorisreduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes.IMPORTANCEThe yeastPichia pastorisnaturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grownP. pastorisin a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found thatP. pastorisdecreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.

scholarly journals The Carbohydrate Metabolism Signature of Lactococcus lactis Strain A12 Reveals Its Sourdough Ecosystem Origin

2013 ◽  
Vol 79 (19) ◽  
pp. 5844-5852 ◽  
Author(s):  
Delphine Passerini ◽  
Michèle Coddeville ◽  
Pascal Le Bourgeois ◽  
Pascal Loubière ◽  
Paul Ritzenthaler ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Lactococcus Lactis ◽  
Specific Growth ◽  
Alternative Pathway ◽  
Soluble Carbohydrates ◽  
Phenotype Microarray ◽  
Content Type ◽  
Survival Capacity ◽  
Lactis Strain ◽  
Specific Growth Rates

ABSTRACTLactococcus lactissubsp.lactisstrain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci.

scholarly journals Isolated Microbial Single Cells and Resulting Micropopulations Grow Faster in Controlled Environments

2012 ◽  
Vol 78 (19) ◽  
pp. 7132-7136 ◽  
Author(s):  
Christian Dusny ◽  
Frederik Sven Ole Fritzsch ◽  
Oliver Frick ◽  
Andreas Schmid
Keyword(s):  
Pichia Pastoris ◽  
Corynebacterium Glutamicum ◽  
Growth Rates ◽  
Specific Growth ◽  
Single Cells ◽  
Hansenula Polymorpha ◽  
Content Type ◽  
Controlled Environments ◽  
Specific Growth Rates ◽  
Extracellular Environment

ABSTRACTSingularized cells ofPichia pastoris,Hansenula polymorpha, andCorynebacterium glutamicumdisplayed specific growth rates under chemically and physically constant conditions that were consistently higher than those obtained in populations. This highlights the importance of single-cell analyses by uncoupling physiology and the extracellular environment, which is now possible using the Envirostat 2.0 concept.

scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

scholarly journals Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0144224 ◽  
Author(s):  
Mauricio Vergara ◽  
Julio Berrios ◽  
Irene Martínez ◽  
Alvaro Díaz-Barrera ◽  
Cristian Acevedo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Rates ◽  
Cho Cells ◽  
Specific Growth ◽  
Mild Hypothermia ◽  
Chemostat Cultures ◽  
Specific Growth Rates

Morphogenetic expression of Arthrobacter globiformis 425 in continuous culture with carbon or biotin limitation

1978 ◽  
Vol 24 (1) ◽  
pp. 28-30 ◽  
Author(s):  
Adrian P. Wills ◽  
E. C. S. Chan
Keyword(s):  
Growth Rate ◽  
Growth Rates ◽  
Specific Growth ◽  
Normal Growth ◽  
Arthrobacter Globiformis ◽  
Glucose Limitation ◽  
Abnormal Morphology ◽  
Chemostat Cultures ◽  
Specific Growth Rates ◽  
Biotin Limitation

When deprived of biotin, Arthrobacter globiformis 425 exhibits abnormal morphology (large, branched forms of variable size) and a retardation of its normal growth rate. In chemostat cultures, when cells were grown under glucose limitation, the morphology was normal (coccoids or rods) at specific growth rates between 0.05 and 0.125 h−1 (doubling times between 14 and 5.5 h, respectively) at 25 °C. The coccoid-to-rod morphogenesis occurs at a specific growth rate of 0.11 h−1. At the same specific growth rates and temperature, but under biotin limitation, abnormal morphology was observed.

scholarly journals The Plasmid Complement of Lactococcus lactis UC509.9 Encodes Multiple Bacteriophage Resistance Systems

2014 ◽  
Vol 80 (14) ◽  
pp. 4341-4349 ◽  
Author(s):  
Stuart Ainsworth ◽  
Jennifer Mahony ◽  
Douwe van Sinderen
Keyword(s):  
Lactococcus Lactis ◽  
Starter Cultures ◽  
Phenotypic Traits ◽  
Abortive Infection ◽  
Content Type ◽  
Bacteriophage Resistance ◽  
Escape Mutants ◽  
Fermented Dairy Products ◽  
Fermented Dairy ◽  
Dairy Starter

ABSTRACTLactococcus lactissubsp.cremorisstrains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin,L. lactisstrains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids ofL. lactisUC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance ofL. lactisUC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations inorf6, which encodes the major capsid protein of this phage.

scholarly journals Molecular Analysis of theIn SituGrowth Rates of Subsurface Geobacter Species

2012 ◽  
Vol 79 (5) ◽  
pp. 1646-1653 ◽  
Author(s):  
Dawn E. Holmes ◽  
Ludovic Giloteaux ◽  
Melissa Barlett ◽  
Milind A. Chavan ◽  
Jessica A. Smith ◽  
...  
Keyword(s):  
Growth Rates ◽  
Ribosomal Proteins ◽  
Specific Growth ◽  
Expression Patterns ◽  
Transcript Abundance ◽  
Metal Reduction ◽  
Content Type ◽  
Specific Growth Rates ◽  
Dissimilatory Metal Reduction

ABSTRACTMolecular tools that can provide an estimate of thein situgrowth rate ofGeobacterspecies could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate ofGeobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins,rpsCandrplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance ofrpsCcorrelated best (r2= 0.90) with specific growth rates. Therefore, expression patterns ofrpsCwere used to estimate specific growth rates ofGeobacterspecies during anin situuranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression ofGeobacter rpsC, and the increase in the number ofGeobactercells estimated with fluorescentin situhybridization compared well with specific growth rates estimated from levels ofin situ rpsCexpression. However, in later phases, cell number increases were substantially lower than predicted fromrpsCtranscript abundance. This change coincided with a bloom of protozoa and increased attachment ofGeobacterspecies to solid phases. These results suggest that monitoringrpsCexpression may better reflect the actual rate thatGeobacterspecies are metabolizing and growing duringin situuranium bioremediation than changes in cell abundance.

scholarly journals Use of a Genetically Enhanced, Pediocin-Producing Starter Culture, Lactococcus lactis subsp. lactis MM217, To Control Listeria monocytogenes in Cheddar Cheese

1998 ◽  
Vol 64 (12) ◽  
pp. 4842-4845 ◽  
Author(s):  
Nurliza Buyong ◽  
Jan Kok ◽  
John B. Luchansky
Keyword(s):  
Listeria Monocytogenes ◽  
Lactococcus Lactis ◽  
Manufacturing Process ◽  
Acid Production ◽  
Starter Culture ◽  
Cheddar Cheese ◽  
Starter Cultures ◽  
Significant Potential ◽  
Ph Values

ABSTRACT Cheddar cheese was prepared with Lactococcus lactissubsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 106 CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 103 CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 107 CFU per g after 2 weeks of ripening and then gradually decreased to about 103 CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 102 CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.

Organic matter reduction using four densities of seaweed (Gracilaria verucosa) and green mussel (Perna viridis) to improve water quality for aquaculture in Java, Indonesia

2021 ◽  
Vol 34 ◽  
pp. 5
Author(s):  
Lestari L. Widowati ◽  
S. Budi Prayitno ◽  
Sri Rejeki ◽  
Tita Elfitasari ◽  
Pujiono W. Purnomo ◽  
...  
Keyword(s):  
Water Quality ◽  
Specific Growth ◽  
Ammonia Nitrogen ◽  
North Coast ◽  
Small Scale ◽  
Perna Viridis ◽  
Quality Of Water ◽  
Total Ammonia ◽  
Specific Growth Rates

The high organic waste content of river water in Demak, north coast of Java, has caused traditional small-scale pond farmers to stop stocking shrimp. This paper examines whether seaweed and mussel will improve the quality of water these farmers use. The effect of Gracilaria verucosa and Perna viridis on the water quality was assessed by measuring the removal rates (RRs) of total organic material (TOM), total ammonia nitrogen (TAN), nitrite, and nitrate. The specific growth rates (SGRs) of seaweed and mussel were also measured. Thirty-six semi-outdoor tanks containing 800 L of brackish water and 7 cm substrate were randomly assigned to four replications of four densities of G. verucosa: 50 (S50), 100 (S100), 150 (S150), and 200 (S200) g m−2, and of P. viridis: 60 (M60), 90 (M90), 120 (M120), and 150 (M150) g m−2. Weekly, the TOM, TAN, nitrite, and nitrate contents were measured, seaweed and mussel weighted; RRs and SGRs were calculated at the end of the study. The effect of densities on the RRs was significant for both seaweed and mussel. P. viridis was more effective in reducing TOM (by 38%) than G. verucosa (7%); G. verucosa achieved higher RRs for TAN, nitrite, and nitrate. At S200, TOM and TAN decreased by 7.4% and 67%, respectively. At M90, TOM and TAN, decreased by 38% and 49%, respectively. However, nitrite increased significantly at S200 and M150. The SGR of seaweed was significantly lower at S200 than that at S150, S100, and S50. The best performing densities were S100 and M90.

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