The Overexpression of YALI0B07117g Results in EnhancedErythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h ) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

Continuous cultivation of the lithoautotrophic nitrate-reducing Fe(II)-oxidizing culture KS in a chemostat bioreactor

Laboratory-based studies on microbial Fe(II) oxidation are commonly performed over just a few weeks in small volumes with high substrate concentrations, resulting in geochemical gradients and volumetric effects caused by sampling. We used a chemostat to enable uninterrupted supply of medium, and investigated autotrophic growth of the nitrate-reducing Fe(II)-oxidizing culture KS for 24 days. We analysed Fe- and N-speciation, cell-mineral associations, and the identity of minerals. Results were compared to different batch systems (50 and 700 ml – static/shaken). The Fe(II) oxidation rate was highest in the chemostat with 7.57 mM Fe(II) d-1, while the extent was similar (averaged 92% of all Fe(II)). Short-range ordered Fe(III) phases, presumably ferrihydrite, precipitated and later goethite was detected in the chemostat. 1 mM solid phase Fe(II) remained in the chemostat, up to 15 µM of reactive nitrite was measured, and 42% of visualized cells were partially or completely mineral-encrusted, likely caused by abiotic oxidation of Fe(II) by nitrite. Despite (partial) encrustation, cells were still viable. Our results show that even with similar oxidation rates as in batch cultures, cultivating Fe(II)-oxidizing microorganisms under continuous conditions reveals mechanistic insights on the role of reactive intermediates for Fe(II) oxidation, mineral formation and cell-mineral interactions.

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L−1 h−1.

The Effect of Various Salinities and Light Intensities on the Growth Performance of Five Locally Isolated Microalgae [Amphidinium carterae, Nephroselmis sp., Tetraselmis sp. (var. red pappas), Asteromonas gracilis and Dunaliella sp.] in Laboratory Batch Cultures

After a 1.5-year screening survey in the lagoons of Western Greece in order to isolate and culture sturdy species of microalgae for aquaculture or other value-added uses, as dictated primarily by satisfactory potential for their mass culture, five species emerged, and their growth was monitored in laboratory conditions. Amphidinium carterae, Nephroselmis sp., Tetraselmis sp. (var. red pappas), Asteromonas gracilis, and Dunaliella sp. were batch cultured using low (20 ppt), sea (40 ppt), and high salinity (50 or 60 or 100 ppt) and in combination with low (2000 lux) and high (8000 lux) intensity illumination. The results exhibited that all these species can be grown adequately in all salinities and with the best growth in terms of maximum cell density, specific growth rate (SGR), and biomass yield (g dry weight/L) at high illumination (8000 lux). The five species examined exhibited different responses in the salinities used, whereby Amphidinium clearly performs best in 20 ppt, far better than 40 ppt, and even more so than 50 ppt. Nephroselmis and Tetraselmis grow almost the same in 20 and 40 ppt and less well in 60 ppt. Asteromonas performs best in 100 ppt, although it can grow quite well in both 40 and 60 ppt. Dunaliella grows equally well in all salinities (20, 40, 60 ppt). Concerning the productivity, assessed as the maximum biomass yield at the end of the culture period, the first rank is occupied by Nephroselmis with ~3.0 g d.w./L, followed by Tetraselmis (2.0 g/L), Dunaliella (1.58 g/L), Amphidinium (1.19 g/L), and Asteromonas (0.7 g/L) with all values recorded at high light (8000 lux).

The Effect of Various Salinities and Light Intensities on the Growth Performance of Five Locally Isolated Microalgae [Amphidinium carterae, Nephroselmis sp., Tetraselmis sp. (var. red pappas), Asteromonas gracilis and Dunaliella sp.] in Laboratory Batch Cultures

After a 1.5 year screening survey in the lagoons of Western Greece in order to isolate and culture sturdy species of microalgae for aquaculture or other value added uses, as dictated primarily by a satisfactory potential for their mass-culture, five species emerged and their growth was monitored in laboratory conditions. Amphidinium carterae, Nephroselmis sp., Tetraselmis sp. (var. red pappas), Asteromonas gracilis and Dunaliella sp. were batch cultured using low (20 ppt), sea (40 ppt) and high salinity (50 or 60 or 100 ppt) and in combination with a low (2000 lux) and high (8000 lux) intensity of illumination. The results exhibited that all these species can be grown adequately in all salinities and with best growth in terms of maximum cell density, specific growth rate (SGR) and biomass yield (g dry weight/L) at high illumination (8000 lux). The five species examined exhibited different responses in the salinities used, Amphidinium clearly does best in 20 ppt far better than 40 ppt and even more than 50 ppt. Nephroselmis and Tetraselmis grow almost the same in 20 and 40 ppt and less well in 60 ppt. Asteromonas does best in 100 ppt although it can grow quite well in both 40 and 60 ppt. Dunaliella grows equally well in all salinities (20-40-60 ppt). Concerning productivity as maximum biomass yield at the end of the culture period, first rank is occupied by Nephroselmis with ~3.0 g d.w./L, followed by Tetraselmis (2.0 g/L), Dunaliella (1.58 g/L), Amphidinium (1.19 g/L) and Asteromonas (0.7 g/L) with all values recorded at high light (8000 lux).

Removal of Toxic Volatile Compounds in Batch Culture Prolongs Stationary Phase and Delays Death of Escherichia coli

The mechanisms controlling entry into and exit from death phase in the bacterial life cycle remain unclear. While bacterial growth studies in batch cultures traditionally focus on the first three phases during incubation, two additional phases, death phase and long-term stationary phase, are less understood. Although there are a number of stressors that arise during long-term batch culture, including nutrient depletion and the accumulation of metabolic toxins such as reactive oxidative species, their roles in cell death are not well-defined. By manipulating environmental conditions of Escherichia coli incubated in long-term batch culture through chemical and mechanical means, we investigated the role of volatile metabolic toxins in modulating the onset of death phase. Here, we demonstrate that with the introduction of substrates with high binding affinities for volatile compounds, toxic byproducts of normal cell metabolism, into the headspace of batch cultures, cells display prolonged stationary phase and delayed entry into death phase. Addition of these substrates allows cultures to maintain a high cell density for hours to days longer than cultures incubated under standard growth conditions. A similar effect is observed when the gaseous headspace in culture flasks is continuously replaced with sterile air, mechanically preventing the accumulation of metabolic byproducts in batch cultures. We establish that toxic compound(s) are produced during exponential phase, demonstrate that buildup of toxic byproducts influence entry into death phase, and present a novel tool for improving high density growth in batch culture that may be used in future research, industrial, or biotechnology applications. IMPORTANCE Bacteria, such as Escherichia coli , are routinely used in the production of biomaterials because of their efficient and sustainable capacity for synthesis of bioproducts. Industrial applications of microbial synthesis typically utilize cells in stationary phase, when cultures have the greatest density of viable cells. By manipulating culture conditions to delay the transition from stationary phase to death phase, we can prolong stationary phase on a scale of hours to days, thereby maintaining the maximum density of cells that would otherwise quickly decline. Characterization of the mechanisms that control entry into death phase for the model organism Escherichia coli not only deepens our understanding of the bacterial life cycle, but also presents an opportunity to enhance current protocols for batch culture growth and explore similar effects in a variety of widely used bacterial strains.

Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability

Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel).

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.