The effects of Pinus brutia bark extract on pure and mixed continuous cultures of rumen bacteria and archaea, and fermentation characteristics in vitro

The aim of the study was to investigate the effects of Pinus brutia bark extract, which is rich in polyphenolic compounds of tannins, on both pure and mixed continuous cultures of rumen bacteria and archaea, as well as on rumen fermentation characteristics in vitro. Antimicrobial susceptibility assay with pure cultures was carried out in an anaerobic chamber. Pinus brutia bark extract exhibited a potential inhibitor activity (P<0.05) against pure cultures of Ruminococcus flavefaciens, Eubacterium ruminantium, and Methanobacterium formicicum while a growth stimulatory effect (P<0.05) was observed for Ruminoccocus albus, Butyrivibrio fibrisolvens, and Streptococcus bovis. Pinus brutia bark extract only had a potential inhibitor effect (P<0.05) on R. albus at the highest dose (1200 µg/mL). Pinus brutia bark extract also stimulated (P<0.05) the growth of pure cultures of Fibrobacter succinogenes, while it did not affect Megasphaera elsdenii, except at the highest dose. The effects of two doses (75 and 375 mg/L) of P. brutia bark extract on in vitro mixed cultures and rumen fermentation parameters were determined by the rumen simulation technique (Rusitec). Supplementation with P. brutia bark extract led to a quadratic decrease (P<0.05) in the cell numbers of R. flavefaciens. Production of total and individual short chain fatty acids (SCFA), acetate to propionate ratio (C2/C3), total protozoa, ruminal pH, and dry matter digestibility (DMD) did not change in the presence of P. brutia bark extract. Supplementation with both doses of P. brutia bark extract decreased (P<0.05) the ammonia-N concentrations. Ammonia-N concentration was lowest in the high-supplemented group (P<0.05). As a conclusion, inhibitory effects of P. brutia bark extract on some species in the pure cultures were in the same direction as with mixed ruminal cultures, while stimulatory effects disappeared. The lack of inhibitory effects on protozoa and on a large number of Gram-positive rumen bacteria in the mixed cultures suggests that its mechanism of action is not exactly similar to antibiotics. Although P. brutia bark extract did not alter ruminal SCFA, it could have potential to improve ruminal protein utilization without depressing rumen microbial fermentation.

Inferring metabolic fluxes in nutrient-limited continuous cultures: A Maximum Entropy Approach with minimum information

We propose a new scheme to infer the metabolic fluxes of cell cultures in a chemostat. Our approach is based on the Maximum Entropy Principle and exploits the understanding of the chemostat dynamics and its connection with the actual metabolism of cells. We show that, in continuous cultures with limiting nutrients, the inference can be done with limited information about the culture: the dilution rate of the chemostat, the concentration in the feed media of the limiting nutrient and the cell concentration at steady state. Also, we remark that our technique provides information, not only about the mean values of the fluxes in the culture, but also its heterogeneity. We first present these results studying a computational model of a chemostat. Having control of this model we can test precisely the quality of the inference, and also unveil the mechanisms behind the success of our approach. Then, we apply our method to E. coli experimental data from the literature and show that it outperforms alternative formulations that rest on a Flux Balance Analysis framework.

A light tunable differentiation system for the creation and control of consortia in yeast

Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, we present an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, our system enables dynamic control of consortia composition in continuous cultures for extended periods. We further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. Our artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.

PAOX1 expression in mixed-substrate continuous cultures of Komagataella phaffii (Pichia pastoris) is completely determined by methanol uptake regardless of the secondary carbon source: a simplifying principle for predicting recombinant protein production.

The expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional "secondary" carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses PAOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and AOX1 over a wide range of dilution rates (0.02-0.20 h-1) in continuous cultures of the Mut+ strain fed with methanol, methanol + glycerol, and methanol + sorbitol. We find that when the specific AOX1 expression and methanol uptake rates for each of the three feeds are plotted against each other, they collapse into a single hyperbolic curve. The specific AOX1 expression rate is therefore completely determined by the specific methanol uptake rate regardless of the existence (present/absent) and type (repressing/non-repressing) of the secondary carbon source. In particular, cultures fed with methanol + glycerol and methanol + sorbitol that consume methanol at equal rates also express the protein at equal rates and levels. Now, it turns out that the simple unstructured model developed by Egli and co-workers can predict the specific methanol uptake rates of single- and mixed-substrate cultures over a wide range of dilution rates and feed concentrations. By combining this model with our data, we derive simple formulas that predicts the protein expression rates and levels of single- and mixed-substrate cultures over a wide range of conditions.

Approaches and involved principles to control pH/pCO2 stability in algal cultures

Experimental cultures of both microalgae and macroalgae are commonly carried out by phycologists or environmental biologists to look into morphological, physiological, and molecular responses to aquatic environmental changes. However, the species of inorganic carbon in algae cultures is often altered by algal photosynthetic CO2 removal and/or bicarbonate utilization. The pH changes associated with altered carbonate chemistry in cultures impact physiological processes in microalgae and macroalgae even at their exponential growth phases, since extra energy is required to sustain intracellular acid–base homeostasis. Usually, pH increases during light period due to inorganic carbon uptake and utilization for photosynthesis and decreases during dark period because of respiratory CO2 release. Therefore, to obtain relevant data aimed for physiological and/or molecular responses of algae to changed levels of environmental factors, stability of pH/pCO2 in the cultures should be considered and controlled to rule out impacts of carbonate chemistry and pH changes. In this work, principles involved in changing pH processes in algal cultures are mechanistically analyzed and several approaches to control pH and pCO2 are introduced. In order to sustain stability of pH/pCO2, the principles underline the following key points: (1) maintaining the rate of photosynthetic C removal less than or equal to the rate of CO2 dissolution into the cultures which are aerated; or (2) sustaining dilute cultures with very low cell density without aeration, so that photosynthetic C removal is small enough not to cause significant pH/pCO2 changes; or (3) stabilizing the changes in micro-environments surrounding the cells or thallus. To maintain pH drift < 1% in growing typical unicellular microalgae, the recommended cell concentration ranges from 50 × 103 to 200 × 103 mL−1 with aeration (air replacement rate of ca 500–1000 mL L−1 min−1) in semi-continuous cultures of < 1 L, and it ranges from 100 to 5000 cells mL−1 for diatoms and from 100 to 100 × 103 cells mL−1 for coccolithophores in dilute cultures without aeration, respectively. For macroalgae, maintaining the thalli in flowing through- system or in semi-continuous cultures (continuously control algal biomass density) is recommended.

A light tunable differentiation system for the creation and control of consortia in yeast

Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, we present an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, our system enables dynamic control of consortia composition in continuous cultures for extended periods. We further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. Our artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.