Growth In Batch Culture Research Articles

A simple interaction-based E. coli growth model

We present a simple growth model which was developed to explain Escherichia coli growth in batch culture. Optical density measurements are used to obtain E. coli growth curves for different inoculum sizes and nutrients concentrations. The model is described by two nonlinear mutually dependent differential equations that describe time evolution of bacteria and nutrients concentration. Introduction of the negative bacterium–bacterium interaction term is specific for the model and leads to the population decay. The proposed model describes entire experimental growth curves. The growth rate, as a function of initial nutrients concentration, follows the Monod function, whilst during the growth it decreases proportionally with the concentration of nutrients. The parameters in our equations can be related to the parameters of the logistic model. The proposed model can be applied to different E. coli strains and, because of the universality of the equations, might be applied to other bacterial strains.

Расчёт удельной скорости роста микроводорослей

В работе рассматриваются методические приёмы количественной оценки удельной скорости роста микроводорослей в периодической и непрерывной культуре. Показано, что для доказательства постоянства удельной скорости роста достаточно неизменности либо соотношения двух химических характеристик биомассы, либо размерной структуры популяции клеток. Проведён критический анализ правомерности использования логарифмической формулы для оценки удельной скорости роста (µ) микроводорослей в экспоненциальной фазе роста накопительной культуры: µ = (lnB2 – lnB1) / (t2 – t1), где В1 и В2 ― плотности (концентрации) культуры в моменты времени t1 и t2 соответственно. Эта формула повсеместно используется большинством исследователей микроводорослей без доказательств экспоненциального характера роста. При наличии таких доказательств применение логарифмической формулы теряет смысл. Приведены примеры количественного описания экспериментальных данных для двух видов морских микроводорослей в экспоненциальной и линейной фазах роста культуры.

Effect of fluoroquinolone resistance mutation Thr-82→Ile on Clostridioides difficile fitness.

Fluoroquinolone resistance is common among epidemic Clostridioides difficile PCR ribotype (RT) 027 and may have contributed to outbreaks of C. difficile infection (CDI). We investigated the impact of fluoroquinolone mutations on the bacterial fitness (BF) of C. difficile RT027 isolates. The BF of seven RT027 mutants with reduced susceptibility to moxifloxacin (moxifloxacin MIC 4-32 mg/L) was compared with their susceptible (moxifloxacin MIC 1-2 mg/L) progenitor strains in competitive batch culture (CBC), cell cytotoxicity and maximal growth rate assays. Comparative fitness dynamics of one gyrA Thr-82→Ile-harbouring isolate (CD3079M) versus the parent strain (CD3079) were also investigated in a continuous co-culture (CC) chemostat model. Mutant and parent strain populations were assessed every 24 h over 8 days using selective and non-selective agars. Sequencing was performed using NEBNext® Ultra™ chemistry and Illumina® HiSeq 3000 technologies. BF was significantly increased in all Thr-82→Ile isolates (w = 1.08-1.22) in CBC assays (P = 0.002). Gly-429→Val and Gln-434→Lys (gyrB) also showed no burden to fitness (w = 1.24 and 1.18, respectively), but Asp-71→Tyr conferred reduced fitness (w = 0.80). CC results for strains CD3079 and CD3079M (Thr-82→Ile) supported CBC findings; mutant-to-parent ratios differed significantly by 96 h (x¯=1.80, P = 0.025). The absence of a fitness cost associated with the most prevalent fluoroquinolone resistance mutations may have contributed to the success of RT027. Furthermore, a demonstrable in vitro advantage over fluoroquinolone-susceptible parent strains in CC may contribute to the maintenance of RT027, even in the absence of fluoroquinolone selection pressure.

Photosynthetic and growth responses of Nannochloropsis oculata (Eustigmatophyceae) during batch cultures in relation to light intensity

:Light is one of the most important factors affecting photosynthesis and growth of microalgal cultures. Photosynthetic responses to light change with the stage of the culture; however, little is known about how changes in the photosynthetic capacity with the duration of batch culture affect growth at the end (final harvest day) of the culture. Accordingly, we studied the photosynthetic performance and growth of Nannochloropsis oculata in the exponential phase (day 4), the linear phase of growth (day 8) and stationary phase (day 15) of batch cultures grown under a range of light intensities from 20 to 200 μmol photons m−2 s−1. Nannochloropsis oculata showed a high ability to acclimate to changing light conditions; cells almost doubled their cellular Chl a concentration under low light conditions and increased their photosynthetic oxygen evolution capacity, which allowed this alga to maintain a stable photosynthetic efficiency in the three stages of the culture studied. Changes in the Chl a per cell under different light conditions were not paralleled by changes in antenna size, which suggests that N. oculata uses an alternative strategy to acclimate to changing light levels. Finally, increasing photosynthetic capacity in cells growing under 20 μmol photons m−2 s−1 did not avoid light limitation and decreasing exponential growth rate. However, the lack of differences in final cell density achieved between 20 and 200 μmol photons m−2 s−1 treatments suggests that bicarbonate limitation started to affect photosynthesis and growth at the higher light treatments, from the linear phase of growth onwards. Understanding of the photosynthetic and growth responses of N. oculata under changing light conditions, where the heterogeneity of light distribution in the cultures substantially affects growth, is crucial to optimisation of the photosynthetic efficiency and growth of microalgal cultures.

The Expression Pattern of the Phosphoproteome Is Significantly Changed During the Growth Phases of Recombinant CHO Cell Culture.

Post-translational modification of proteins by reversible phosphorylation plays a pivotal role in regulating key cellular processes including transcription, translation, cell proliferation, differentiation, apoptosis, and signal transduction. Despite the importance of the phosphorylation level of regulation, little work has been carried out on the phosphoproteomic characterization of Chinese hamster ovary (CHO) cells in bioprocess-relevant conditions. Growth control strategies are often used to prolong culture duration and increase specific productivity; however, the cellular mechanisms and regulatory pathways underlying growth strategies are poorly understood in CHO cells. Phosphorylation changes are dynamic and will respond to changes in culture conditions; this may reflect the status of the cells with respect to growth and viability of the culture. Herein, this study uses a phosphopeptide enrichment strategy in conjunction with LC-MS/MS to carry out a large-scale differential phosphoproteomic analysis of IgG producing CHO DP12 cells at various phases of growth in serum-free suspension batch culture to characterize dynamic changes to the phosphoproteome with changing culture conditions. In total over the various growth phases, 3777 differentially expressed unique phosphopeptides are identified from 1415 differentially expressed unique phosphoproteins. Analysis of the whole cell lysate without phosphopeptide enrichment over the various growth phases revealed the differential expression of 834 unique proteins, with an overlap of 188 proteins between the proteomic and phosphoproteomic analyses. The inclusion of phosphoproteomic data significantly improves proteome coverage but also gives insights into the post-translational level of regulation during cellular growth of recombinant CHO cells.

Differential carbon partitioning and fatty acid composition in mixotrophic and autotrophic cultures of a new marine isolate Tetraselmis sp. KY114885

Biochemical composition of microalgae, a promising feedstock for biodiesel production, can be modified under different nutritional modes. The objective of this study was to investigate the influence of autotrophy and mixotrophy on the growth and biochemical composition of Tetraselmis sp. KY114885. Time-course variations of lipids and starch were determined using gravimetric and colorimetric methods. After direct transesterification, lipid profile on days 12 and 20 of cultivation period was determined. The results revealed higher growth of microalgae in mixotrophic cultures containing 5 g L−1 glucose, whereas 7.5 g L−1 glucose suppressed growth. Mixotrophy enhanced the lipid and starch yields in a distinctive trend. The fatty acid composition changed with the different nutritional regimes and the length of growth in batch culture. Glucose supplementation elevated the portions of monounsaturated fatty acids, particularly oleic acid, and decreased the percentage of saturated and polyunsaturated fatty acids. The present study suggests that mixotrophic cultivation of Tetraselmis sp. KY114885 may be a feasible strategy to improve growth parameters and the biochemical composition of the alga for biofuel production. The results also shed light on carbon allocation in the algae grown under two different trophic modes.

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast.

Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.

Robust Mercury Methylation across Diverse Methanogenic Archaea.

ABSTRACTMethylmercury (MeHg) production was compared among nine cultured methanogenic archaea that contain hgcAB, a gene pair that codes for mercury (Hg) methylation. The methanogens tested produced MeHg at inherently different rates, even when normalized to growth rate and Hg availability. Eight of the nine tested were capable of MeHg production greater than that of spent- and uninoculated-medium controls during batch culture growth. Methanococcoides methylutens, an hgcAB+ strain with a fused gene pair, was unable to produce more MeHg than controls. Maximal conversion of Hg to MeHg through a full batch culture growth cycle for each species (except M. methylutens) ranged from 2 to >50% of the added Hg(II) or between 0.2 and 17 pmol of MeHg/mg of protein. Three of the species produced >10% MeHg. The ability to produce MeHg was confirmed in several hgcAB+ methanogens that had not previously been tested (Methanocella paludicola SANAE, Methanocorpusculum bavaricum, Methanofollis liminatans GKZPZ, and Methanosphaerula palustris E1-9c). Maximal methylation was observed at low sulfide concentrations (<100 μM) and in the presence of 0.5 to 5 mM cysteine. For M. hollandica, the addition of up to 5 mM cysteine enhanced MeHg production and cell growth in a concentration-dependent manner. As observed for bacterial Hg methylators, sulfide inhibited MeHg production. An initial evaluation of sulfide and thiol impacts on bioavailability showed methanogens responding to Hg complexation in the same way as do Deltaproteobacteria. The mercury methylation rates of several methanogens rival those of the better-studied Hg-methylating sulfate- and iron-reducing Deltaproteobacteria.

Prodigiosin pigment of Serratia marcescens is associated with increased biomass production.

Serratia marcescens is a gram-negative, facultatively-anaerobic bacterium and opportunistic pathogen which produces the red pigment prodigiosin. We employed both batch culture and chemostat growth methods to investigate prodigiosin function in the producing organism. Pigmentation correlated with an increased rate of ATP production during population lag phase. Results with a lacZ transcriptional fusion to the prodigiosin (pig) biosynthetic operon revealed that operon transcription is activated by low cellular levels of ATP at high cell density. Furthermore, these data enabled estimation of the ATP per cell minimum value at which the operon is induced. Pigmented cells were found to accumulate ATP more rapidly and to multiply more quickly than non-pigmented cells during the high density growth phase. Finally, results with both batch and chemostat culture revealed that pigmented cells grow to approximately twice the biomass yield as non-pigmented S. marcescens bacteria. Prodigiosin production may, therefore, provide a growth advantage at ambient temperatures.

Continuous culture techniques as simulators for standard cells: Jacques Monod's, Aron Novick's and Leo Szilard's quantitative approach to microbiology.

Continuous culture techniques were developed in the early twentieth century to replace cumbersome studies of cell growth in batch cultures. In contrast to batch cultures, they constituted an open concept, as cells are forced to proliferate by adding new medium while cell suspension is constantly removed. During the 1940s and 1950s new devices have been designed-called "automatic syringe mechanism," "turbidostat," "chemostat," "bactogen," and "microbial auxanometer"-which allowed increasingly accurate quantitative measurements of bacterial growth. With these devices cell growth came under the external control of the experimenters and thus accessible for developing a mathematical theory of growth kinetics-developed mainly by Jacques Monod, Aron Novick and Leo Szilard in the early 1950s and still in use today. The paper explores the development of continuous culture devices and claims that these devices are simulators for standard cells following specific requirements, in particular involving mathematical constraints in the design and setting of the devices as well as experiments. These requirements have led to contemporary designs of continuous culture techniques realizing a specific event-based flow algorithm able to simulate directed evolution and produce artificial cells and microorganisms. This current development is seen as an alternative approach to today's synthetic biology.

Methods for Generating Microbial Cocultures that Grow in the Absence of Fixed Carbon or Nitrogen.

This work is motivated by both (1) the desire to make interesting products without the reliance on fixed carbon or fixed nitrogen using microbial cocultures, and (2) the desire to develop new methods for growing microbial communities. The bioplastic polyhydroxybutyrate (PHB), for example, is considered prohibitively expensive to make from sugar (compared to polypropylene). Utilizing and building on engineered strains of Synechococcus elongatus and Azotobacter vinelandii, we have combined a nitrogen-fixing organism with a carbon-fixing organism to make PHB from air, water, sunlight, and trace minerals. Our observations of coculture growth in batch culture led us to develop an improved system based on manipulating the osmotic pressure within a hydrogel. The methods we used to develop this coculture are described in detail in the following chapter, including notes detailing some of our additional observations or thoughts on this system.

Assessing Carbon Source-Dependent Phenotypic Variability in Pseudomonas putida.

The soil bacterium Pseudomonas putida is rapidly becoming a platform of choice for applications that require a microbial host highly resistant to different types of stresses and elevated rates of reducing power regeneration. P. putida is capable of growing in a wide variety of carbon sources that range from simple sugars to complex substrates such as aromatic compounds. Interestingly, the growth of the reference strain KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. This macroscopic phenomenon has been shown to be connected with the stochastic expression of the glp genes, which encode the enzymes needed for glycerol processing. In this protocol, we propose a general procedure to examine bacterial growth in small-scale cultures while monitoring the metabolic activity of individual cells. Assessing the metabolic capacity of single bacteria by means of fluorescence microscopy and flow cytometry, in combination with the analysis of the temporal takeoff of growth in single-cell cultures, is a simple and easy-to-implement approach. It can help to understand the link between macroscopic phenotypes (e.g., microbial growth in batch cultures) and stochastic phenomena at the genetic level. The implementation of these methodologies revealed that the adoption of a glycerol-metabolizing regime by P. putida KT2440 is not the result of a gradual change in the whole population, but it rather reflects a time-dependent bimodal switch between metabolically inactive (i.e., not growing) to fully active (i.e., growing) bacteria.

HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp

Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labelling with 32P-labelled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40- and ≥8-fold, respectively) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.

Sensitivity of nonfermentative gram-negative bacteria to essential oils of different origin

The sensitivity of MDR (multi drug resistant) strains of Stenotrophomonas maltophilia, Acinetobacter baumannii, and Pseudomonas aeruginosa to essential oils and their individual components was studied; bacteriostatic and bactericidal concentrations of 16 substances were determined. Crimean rose oil exhibited the highest activity, with the minimum inhibitory concentration of 1.95 μL/ mL. Growth of bacterial batch cultures in the presence of subinhibitory concentrations of essential oils or their individual components was studied. Kinetic models analysis revealed positive correlations of growth characteristics of the studied bacteria the effects of essential oils (p-level < 0.05). Correlations between lag phase duration and the death of bacterial cultures and correlations between the concentration of linalool (a component of essential oils) and the degree of growth suppression for S. maltophilia and A. baumannii were revealed.

Absolute noninvasive measurement of CO2 mole fraction emitted by E. coli and S. aureus using calibration-free 2f WMS applied to a 2004 nm VCSEL.

We report the first demonstration, to the best of our knowledge, of accurate real-time noninvasive measurement of the absolute cumulative mole fraction of metabolic carbon dioxide emitted by Escherichia coli and Staphylococcus aureus over a period of several hours of their life cycles using a recently developed calibration-free wavelength modulation spectroscopy technique. A 1mW vertical-cavity surface-emitting laser is used to interrogate a single rotational vibrational absorption line of carbon dioxide at 2003.5nm. The measurements are immune to laser intensity fluctuations and variable optical coupling that is inevitable in such free-space coupled experiments that run over 10-18h. The cumulative carbon dioxide mole fraction follows the characteristic modified Gompertz model that is typical of bacterial growth in batch cultures. The characteristic growth parameters are extracted from this curve. The technique can be readily extended to study multiple volatile organic compounds that bacteria are known to emit.

Aerobic Dechlorination of Dichloromethane Using Biostimulation Agent BD-C in Continuous and Batch Cultures of Xanthobacter autotrophicus GJ10.

It is important to construct microbiological treatment systems for organic solvent-contaminated water. We developed a continuous culture supplemented with a biostimulation agent named BD-C, which is formulated from canola oil, and Xanthobacter autotrophicus strain GJ10 for an aerobic dichloromethane (DCM)-dechlorinating microorganism. The continuous culture was a chemostat constructed using a 1 L screw-capped bottle containing artificial wastewater medium with 2.0 mM DCM and 1.0% (v/v) BD-C. The expression of genes for DCM metabolism in the dechlorinating aerobe was monitored and analyzed by reverse transcription-quantitative PCR. Strain GJ10 was able to dechlorinate approximately 74% of the DCM in medium supplemented with BD-C during 12 days of incubation. The DCM dechlorination rate was calculated to be 0.11 mM/day. The ΔΔCT method showed that expression of haloalkane dehalogenase increased 5.4 times in the presence of BD-C. Based on batch culture growth tests conducted with mineral salt medium containing three DCM concentrations (0.07, 0.20, 0.43 and 0.65 mM) with BD-C, the apparent maximum specific consumption rate (νmax) and the saturation constant (Ks) determined for DCM degradation in this test were 19.0 nmol/h/CFU and 0.44 mM, respectively. In conclusion, BD-C enhanced the aerobic degradation of DCM by strain GJ10.

Photosynthetic efficiency predicts toxic effects of metal nanomaterials in phytoplankton

High Throughput Screening (HTS) using in vitro assessments at the subcellular level has great promise for screening new chemicals and emerging contaminants to identify high-risk candidates, but their linkage to ecological impacts has seldom been evaluated. We tested whether a battery of subcellular HTS tests could be used to accurately predict population-level effects of engineered metal nanoparticles (ENPs) on marine phytoplankton, important primary producers that support oceanic food webs. To overcome well-known difficulties of estimating ecologically meaningful toxicity parameters, we used novel Dynamic Energy Budget and Toxicodynamic (DEBtox) modeling techniques to evaluate impacts of ENPs on population growth rates. Our results show that population growth was negatively impacted by all four ENPs tested, but the HTS tests assessing many cell/physiological functions lacked predictive power at the population level. However, declining photosynthetic efficiency, a traditional physiological endpoint for photoautotrophs, was a good predictor of population level effects in phytoplankton. DEBtox techniques provided robust estimates of EC10 for population growth rates in exponentially growing batch cultures of phytoplankton, and should be widely useful for ecotoxicological testing. Adoption of HTS approaches for ecotoxicological assessment should carefully evaluate the predictive power of specific assays to minimize the risk that effects at higher levels of biological organization may go undetected.

Transcriptional Control in Marine Copiotrophic and Oligotrophic Bacteria with Streamlined Genomes.

Bacteria often respond to environmental stimuli using transcriptional control, but this may not be the case for marine bacteria such as "Candidatus Pelagibacter ubique," a cultivated representative of the SAR11 clade, the most abundant organism in the ocean. This bacterium has a small, streamlined genome and an unusually low number of transcriptional regulators, suggesting that transcriptional control is low in Pelagibacter and limits its response to environmental conditions. Transcriptome sequencing during batch culture growth revealed that only 0.1% of protein-encoding genes appear to be under transcriptional control in Pelagibacter and in another oligotroph (SAR92) whereas >10% of genes were under transcriptional control in the copiotrophs Polaribacter sp. strain MED152 and Ruegeria pomeroyi When growth levels changed, transcript levels remained steady in Pelagibacter and SAR92 but shifted in MED152 and R. pomeroyi Transcript abundances per cell, determined using an internal RNA sequencing standard, were low (<1 transcript per cell) for all but a few of the most highly transcribed genes in all four taxa, and there was no correlation between transcript abundances per cell and shifts in the levels of transcription. These results suggest that low transcriptional control contributes to the success of Pelagibacter and possibly other oligotrophic microbes that dominate microbial communities in the oceans. Diverse heterotrophic bacteria drive biogeochemical cycling in the ocean. The most abundant types of marine bacteria are oligotrophs with small, streamlined genomes. The metabolic controls that regulate the response of oligotrophic bacteria to environmental conditions remain unclear. Our results reveal that transcriptional control is lower in marine oligotrophic bacteria than in marine copiotrophic bacteria. Although responses of bacteria to environmental conditions are commonly regulated at the level of transcription, metabolism in the most abundant bacteria in the ocean appears to be regulated by other mechanisms.

Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18–24 nt and 71–252 nt, respectively), genome copy number (3–1,459), and the number of genes targeted (2–107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

Unexpected capacity for organic carbon assimilation by Thermosynechococcus elongatus, a crucial photosynthetic model organism

Genetic modification of key residues of photosystems is essential to identify functionally crucial processes by spectroscopic and crystallographic investigation; the required protein stability favours use of thermophilic species. The currently unique thermophilic photosynthetic model organism is the cyanobacterial genus Thermosynechococcus. We report the ability of Thermosynechococcus elongatus to assimilate organic carbon, specifically D-fructose. Growth in the presence of a photosynthesis inhibitor opens the door towards crucial amino acid substitutions in photosystems by the rescue of otherwise lethal mutations. Yet depression of batch-culture growth after 7 days implies that additional developments are needed.