Environmental conditions during glycerol bioconversion affect 3-hydroxypropionic acid bioproduction by Limosilactobacillus reuteri DSM 17938.

3-Hydroxypropionic acid (3-HP) is a platform molecule whose biological production was carried out by the bacterium Limosilactobacillus reuteri according to a two-step process: first, a growth phase in batch mode on glucose, then a glycerol bioconversion into 3-HP in fed-batch mode. With the objective of improving 3-HP bioproduction, this study aimed at defining the operating conditions during the bioconversion phase that increases the bioproduction performance. A central composite rotatable design allowed testing various pH levels and specific glycerol feeding rates. By establishing response surfaces, optimal conditions have been identified that were different depending on the considered output variable (final 3-HP quantity, 3-HP production yield and production rate). Of them, 3-HP final quantity and 3-HP production yield were maximized at pH 6.0 and at specific glycerol feeding rates of 60 and 55 mggly  gCDW -1  h-1 , respectively. The specific 3-HP production rate was the highest at the upper limit of the specific substrate feeding rate (80 mggly  gCDW -1  h-1 ) but was not affected by the pH. An additional experiment was carried out at pH 6.0 and a specific glycerol feeding rate of 80 mggly  gCDW -1  h-1 to validate the previous observations. In conclusion, the results showed a significant improvement of 3-HP concentration by 13%, of specific production rate by 34% and of 3-HP volumetric productivity by 39%, as compared to the initial values.

A rapid method for the assessment of the vitality of microorganisms using flow cytometry.

Cultivability, viability, and vitality make it possible to characterize the behavior of a cellular population. Vitality was assessed using the kinetic parameters of specific metabolisms depending on whether the strains were used, for example, for the acidification of lactic acid bacteria or for CO2 production in fermenting yeasts. However, these methods are time-consuming. We developed a cytometric descriptor based on the energy-dependent extrusion of carboxyfluorescein from cells, subsequent to carboxyfluorescein diacetate staining, and compared it to the measurements of metabolic activities of various bacteria and yeasts. For all of the microorganisms tested, the cytometric descriptor ΔFI15 was well correlated with the results of the metabolic measurements and, moreover, has the advantage of being easier and faster to use than metabolic methods. It can be very useful for evaluating the vitality of the starters before inoculation in industrial processes.

Efficient 3-hydroxypropionic acid production by Acetobacter sp. CIP 58.66 through a feeding strategy based on pH control.

Acetic acid bacteria (AAB) can selectively oxidize diols into their corresponding hydroxyacids. Notably, they can convert 1,3-propanediol (1,3-PDO) into 3-hydroxypropionic acid (3-HP), which is a promising building-block. Until now, 3-HP production with AAB is carried out in batch and using resting cells at high cell densities (up to 10 g L-1 of cell dry weight). This approach is likely limited by detrimental accumulation of the intermediate 3-hydroxypropanal (3-HPA). Herein, we investigate an alternative implementation that allows highly efficient 3-HP production with lower cell densities of growing cells and that prevents 3-HPA accumulation. First, growth and 3-HP production of Acetobacter sp. CIP 58.66 were characterized with 1,3-PDO or glycerol as growth substrate. The strain was then implemented in a bioreactor, during a sequential process where it was first cultivated on glycerol, then the precursor 1,3-PDO was continuously supplied at a varying rate, easily controlled by the pH control. Different pH set points were tested (5.0, 4.5, and 4.0). This approach used the natural resistance of acetic acid bacteria to acidic conditions. Surprisingly, when pH was controlled at 5.0, the performances achieved in terms of titer (69.76 g3-HP L-1), mean productivity (2.80 g3-HP L-1 h-1), and molar yield (1.02 mol3-HP mol-11,3-PDO) were comparable to results obtained with genetically improved strains at neutral pH. The present results were obtained with comparatively lower cell densities (from 0.88 to 2.08 g L-1) than previously reported. This feeding strategy could be well-suited for future scale-up, since lower cell densities imply lower process costs and energy needs.

Culture conditions affect Lactobacillus reuteri DSM 17938 ability to perform glycerol bioconversion into 3-hydroxypropionic acid.

The platform molecule 3-hydroxypropionic acid (3-HP) can be produced using Lactobacillus reuteri through a two-step bioprocess that involves a growth phase followed by a bioconversion phase. The bioproduction is performed by resting cells that convert glycerol into 3-HP and 1,3-propanediol in fed-batch mode. This work aimed at studying the effect of the growth conditions of L. reuteri DSM 17938 during the first step, on the glycerol bioconversion into 3-HP during the second step. A Plackett and Burman design was carried out to test, in controlled bioreactors, the effect of 11 growth conditions simultaneously, at fixed bioconversion conditions. The supplementation of the growth medium with vitamin B12 and cysteine displayed a negative effect on the 3-HP bioproduction. The addition of glucose, phytone peptone, Tween 80, 1,2-propanediol and betaine in the growth medium, together with a low temperature and an optimal pH of 6.0 during the growth phase increased the bioconversion duration from 56 h to 89 h at a glycerol feeding rate of 0.5 g·h-1. A validating experiment displayed that the 3-HP titer, 3-HP production yield and 3-HP specific production rate were significantly improved by 25 %, 150 % and 61 %, respectively.

Process engineering for microbial production of 3-hydroxypropionic acid.

Due to concerns about the unsustainability and predictable shortage of fossil feedstocks, research efforts are currently being made to develop new processes for production of commodities using alternative feedstocks. 3-Hydroxypropionic acid (CAS 503-66-2) was recognised by the US Department of Energy as one of the most promising value-added chemicals that can be obtained from biomass. This article aims at reviewing the various strategies implemented thus far for 3-hydroxypropionic acid bioproduction. Special attention is given here to process engineering issues. The variety of possible metabolic pathways is also described in order to highlight how process design can be guided by their understanding. The most recent advances are described here in order to draw up a panorama of microbial 3-hydroxypropionic acid production: best performances to date, remaining hurdles and foreseeable developments. Important milestones have been achieved, and process metrics are getting closer to commercial relevance. New strategies are continuously being developed that involve new microbial strains, new technologies, or new carbon sources in order to overcome the various hurdles inherent to the different microbial routes.

Conversion of Glycerol to 3-Hydroxypropanoic Acid by Genetically Engineered Bacillus subtilis.

3-Hydroxypropanoic acid (3-HP) is an important biomass-derivable platform chemical that can be converted into a number of industrially relevant compounds. There have been several attempts to produce 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from K. pneumoniae. Genetic engineering, driven by in silico optimization, and optimization of cultivation conditions resulted in a 3-HP titer of 10 g/L, in a standard batch cultivation. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis. With this relatively high titer in batch, and the robustness of B. subtilis in high density fermentation conditions, we expect that our production strains may constitute a solid basis for commercial production of 3-HP.

Escherichia coli under Ionic Silver Stress: An Integrative Approach to Explore Transcriptional, Physiological and Biochemical Responses.

For a better understanding of the systemic effect of sub-lethal micromolar concentrations of ionic silver on Escherichia coli, we performed a multi-level characterization of cells under Ag+-mediated stress using an integrative biology approach combining physiological, biochemical and transcriptomic data. Physiological parameters, namely bacterial growth and survival after Ag+ exposure, were first quantified and related to the accumulation of intracellular silver, probed for the first time by nano secondary ion mass spectroscopy at sub-micrometer lateral resolution. Modifications in E. coli biochemical composition were evaluated under Ag+-mediated stress by in situ synchrotron Fourier-transform infrared microspectroscopy and a comprehensive transcriptome response was also determined. Using multivariate statistics, correlations between the physiological parameters, the extracellular concentration of AgNO3 and the intracellular silver content, gene expression profiles and micro-spectroscopic data were investigated. We identified Ag+-dependent regulation of gene expression required for growth (e.g. transporter genes, transcriptional regulators, ribosomal proteins), for ionic silver transport and detoxification (e.g. copA, cueO, mgtA, nhaR) and for coping with various types of stress (dnaK, pspA, metA,R, oxidoreductase genes). The silver-induced shortening of the acyl chain of fatty acids, mostly encountered in cell membrane, was highlighted by microspectroscopy and correlated with the down-regulated expression of genes involved in fatty acid transport (fadL) and synthesis/modification of lipid A (lpxA and arnA). The increase in the disordered secondary structure of proteins in the presence of Ag+ was assessed through the conformational shift shown for amides I and II, and further correlated with the up-regulated expression of peptidase (hfq) and chaperone (dnaJ), and regulation of transpeptidase expression (ycfS and ycbB). Interestingly, as these transpeptidases act on the structural integrity of the cell wall, regulation of their expression may explain the morphological damage reported under Ag+-mediated stress. This result clearly demonstrates that the cell membrane is a key target of ionic silver.

Relationships between the use of Embden Meyerhof pathway (EMP) or Phosphoketolase pathway (PKP) and lactate production capabilities of diverse Lactobacillus reuteri strains.

The aims of this study is to compare the growth and glucose metabolism of three Lactobacillus reuteri strains (i.e. DSM 20016, DSM 17938, and ATCC 53608) which are lactic acid bacteria of interest used for diverse applications such as probiotics implying the production of biomass, or for the production of valuable chemicals (3-hydroxypropionaldehyde, 3-hydroxypropionic acid, 1,3-propanediol). However, the physiological diversity inside the species, even for basic metabolisms, like its capacity of acidification or glucose metabolism, has not been studied yet. In the present work, the growth and metabolism of three strains representative of the species diversity have been studied in batch mode. The strains were compared through characterization of growth kinetics and evaluation of acidification kinetics, substrate consumption and product formation. The results showed significant differences between the three strains which may be explained, at least in part, by variations in the distribution of carbon source between two glycolytic pathways during the bacterial growth: the phosphoketolase or heterolactic pathway (PKP) and the Embden-Meyerhof pathway (EMP). It was also shown that, in the context of obtaining a large amount of biomass, DSM 20016 and DSM 17938 strains were the most effective in terms of growth kinetics. The DSM 17938 strain, which shows the more significant metabolic shift from EMP to PKP when the pH decreases, is more effective for lactate production.