Turbidostat

Anaerobic gut fungi (AGF) have potential to valorize lignocellulosic biomass owing to their diverse repertoire of carbohydrate-active enzymes (CAZymes). However, AGF metabolism is poorly understood, and no stable genetic tools are available to manipulate growth and metabolic flux to enhance production of specific targets, e.g., cells, CAZymes, or metabolites. Herein, a cost-effective, Arduino-based, continuous-flow anaerobic bioreactor with online optical density control is presented to probe metabolism and predictably tune fluxes in Caecomyces churrovis...

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays...

In this paper, a stochastic turbidostat model with controllable output is established by using piecewise constant delayed measurements of the substrate concentration. We commence by proving the existence and uniqueness of the global positive solution of the stochastic delayed model. Then, sufficient conditions of extinction and stochastic strong permanence of the biomass are acquired. In quick succession, we investigate the stochastic asymptotical stability of the washout equilibrium as well as the asymptotic behavior of the random paths approaching the interior equilibrium of its corresponding deterministic model by employing the method of Lyapunov functionals...

After light, temperature is the most relevant environmental parameter in outdoors cultivation of microalgae. Suboptimal and supraoptimal temperatures negatively impact growth and photosynthetic performance with a subsequent effect on lipid accumulation. It is generally recognised that lower temperatures trigger an increase in fatty acid desaturation while higher temperatures trigger the opposite reaction. The effect of temperature on lipid classes has been less studied in microalgae and in certain cases, the effect of light cannot be completely excluded...

Hydrothermal liquefaction (HTL) produces bio-crude oil from wet algae along with an aqueous phase (AP). This effluent contains minerals that can be reused for cultivating new microalgae but whose utility remains limited due to the presence of inhibitors. Reduced photosynthetic performance, growth, and null lipid accumulation were observed in wild-type Chlorella vulgaris NIES 227 cultivated in AP (1/200). Adaptive laboratory evolution was studied by batch transfers and turbidostat mode. Both methods effectively counterbalanced AP toxicity and restored the fitness of the microalgae...

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO2 as sole carbon sources. Labeling with 13 C-formate proved the assimilation of the C1 substrate via the pathway metabolites...

The photoautotrophic, unicellular N2 -fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement...

Decoupling growth from product synthesis is a promising strategy to increase carbon partitioning and maximize productivity in cell factories. However, reduction in both substrate uptake rate and metabolic activity in the production phase are an underlying problem for upscaling. Here, we used CRISPR interference to repress growth in lactate-producing Synechocystis sp. PCC 6803. Carbon partitioning to lactate in the production phase exceeded 90%, but CO2 uptake was severely reduced compared to uptake during the growth phase...

Microalgae offer a promising source of biofuel and a wide array of high-value biomolecules. Large-scale cultivation of microalgae at low density poses a significant challenge in terms of water management. High-density microalgae cultivation, however, can be challenging due to biochemical changes associated with growth dynamics. Therefore, there is a need for a biomarker that can predict the optimum density for high biomass cultivation. A locally isolated microalga Cyanobacterium aponinum CCC734 was grown with optimized nitrogen and phosphorus in the ratio of 12:1 for sustained high biomass productivity...

Tisochrysis lutea is an important microalgal species for fucoxanthin and docosahexaenoic acid (DHA) production with an optimum cultivation temperature of approximately 30 °C. The aim of the present work was to develop a winter strain with high productivity at 15 °C. The response of the original strain to a decrease in temperature from 30 °C to 15 °C was investigated in continuous turbidostat experiments. This was followed by adaptation for >180 days at 15 °C and 2 rounds of sorting for cells with high chlorophyll fluorescence (top 5%) using fluorescence-activated cell sorting (FACS)...

SOS box of the recA promoter, PVRecA from Vibrio natriegens was characterized, cloned and expressed in a probiotic strain E. coli Nissle 1917. This promoter was then rationally engineered according to predicted interactions between LexA repressor and PVRecA . The redesigned PVRecA-AT promoter showed a sensitive and robust response to DNA damage induced by UV and genotoxic compounds. Rational design of PVRecA coupled to an amplification gene circuit increased circuit output amplitude 4.3-fold in response to a DNA damaging compound mitomycin C...

Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana . Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing...

Antibiotic resistance frequently evolves through fitness trade-offs in which the genetic alterations that confer resistance to a drug can also cause growth defects in resistant cells. Here, through experimental evolution in a microfluidics-based turbidostat, we demonstrate that antibiotic-resistant cells can be efficiently inhibited by amplifying the fitness costs associated with drug-resistance evolution. Using tavaborole-resistant Escherichia coli as a model, we show that genetic mutations in leucyl-tRNA synthetase (that underlie tavaborole resistance) make resistant cells intolerant to norvaline, a chemical analog of leucine that is mistakenly used by tavaborole-resistant cells for protein synthesis...

Background: Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase ( mtlD ) and mannitol-1-phosphatase ( m1p , in short: a 'mannitol cassette'), proved to be genetically unstable...

Microbial oils are lipids produced by oleaginous microorganisms, which can be used as a potential feedstock for oleochemical production. The oleaginous yeast Rhodotorula toruloides can co-produce microbial oils and high-value compounds from low-cost substrates, such as xylose and acetic acid (from hemicellulosic hydrolysates) and raw glycerol (a byproduct of biodiesel production). One step towards economic viability is identifying the best conditions for lipid production, primarily the most suitable carbon-to-nitrogen ratio (C/N)...

Coadaptation between bacterial hosts and plasmids frequently results in adaptive changes restricted exclusively to host genome leaving plasmids unchanged. To better understand this remarkable stability we transformed naïve E. coli cells with a plasmid carrying an antibiotic-resistance gene and forced them to adapt in a turbidostat environment. We then drew population samples at regular intervals and subjected them to duplex sequencing-a technique specifically designed for identification of low frequency mutations...

The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol...

We present an accessible, robust continuous-culture turbidostat system that greatly facilitates the generation and phenotypic analysis of highly complex libraries in yeast and bacteria. Our system has many applications in genomics and systems biology; here, we demonstrate three of these uses. We first measure how the growth rate of budding yeast responds to limiting nitrogen at steady state and in a dynamically varying environment. We also demonstrate the direct selection of a diverse, genome-scale protein fusion library in liquid culture...

In this contribution, the dynamic behaviors of a turbidostat model with Contois growth rate and delay are investigated. The qualitative properties of the system are carried out including the stability of the equilibria and the bifurcations. More concretely, we exhibit the transcritical bifurcation by reducing the system without delay to a 1-dimensional system on a center manifold and find that Hopf bifurcation occurs by choosing the delay as bifurcation parameter. Also, using the normal form theory and the center manifold theorem we determine the direction and stability of the bifurcating periodic solutions induced by the Hopf bifurcation...

Photoautotrophic growth of Synechocystis sp. PCC 6803 in a flat-panel photobioreactor, run in turbidostat mode under increasing intensities of orange-red light (636 nm), showed a maximal growth rate (0.12 h-1 ) at 300 μmolphotons m-2 s-1 , whereas first signs of photoinhibition were detected above 800 μmolphotons m-2 s-1 . To investigate the dynamic modulation of the thylakoid proteome in response to photoinhibitory light intensities, quantitative proteomics analyses by SWATH mass spectrometry were performed by comparing thylakoid membranes extracted from Synechocystis grown under low-intensity illumination (i...