Two-stage Fermentation Process Research Articles

Characteristics of whole wheat steamed bread production by two-stage fermentation procedure with Jiaozi as starter

Given the beneficial role of biological acidification in improving the quality of cereal-based products, this study evaluated the applicability of Jiaozi as a starter for whole wheat dough fermentation and steamed bread quality improvement. During the first 12 h of dough fermentation, yeast and lactic acid bacteria (LAB) populations increased significantly, resulting in rapid carbohydrate consumption and acidification of the dough. The lactic/acetic acid molar ratio was approximately 3.3, which was within the recommended fermentation quotient (FQ). Compared with the unfermented dough and the dough fermented for 12 h, the dough fermented for 6 h had an improved protein network structure, and the steamed bread made from it also showed the best quality. At a low leavening temperature of 28 °C, satisfactory specific volume and height/diameter ratio were obtained. The dough with an inoculation amount of 50% of the fully fermented dough showed the highest CO2 production capacity. Compared with yeast, the whole wheat steamed bread produced by Jiaozi using the two-stage fermentation process showed higher specific volume, resistant starch (RS) content and protein digestibility. The results showed that the use of Jiaozi as a starter has great potential for application in improving the quality of whole wheat steamed bread.

Lactate-mediated medium-chain fatty acid production from expired dairy and beverage waste

Fruits, vegetables, and dairy products are typically the primary sources of household food waste. Currently, anaerobic digestion is the most used bioprocess for the treatment of food waste with concomitant generation of biogas. However, to achieve a circular carbon economy, the organics in food waste should be converted to new chemicals with higher value than energy. Here we demonstrate the feasibility of medium-chain carboxylic acid (MCCA) production from expired dairy and beverage waste via a chain elongation platform mediated by lactate. In a two-stage fermentation process, the first stage with optimized operational conditions, including varying temperatures and organic loading rates, transformed expired dairy and beverage waste into lactate at a concentration higher than 900 mM C at 43 °C. This lactate was then used to produce >500 mM C caproate and >300 mM C butyrate via microbial chain elongation. Predominantly, lactate-producing microbes such as Lactobacillus and Lacticaseibacillus were regulated by temperature and could be highly enriched under mesophilic conditions in the first-stage reactor. In the second-stage chain elongation reactor, the dominating microbes were primarily from the genera Megasphaera and Caproiciproducens, shaped by varying feed and inoculum sources. Co-occurrence network analysis revealed positive correlations among species from the genera Caproiciproducens, Ruminococcus, and CAG-352, as well as Megasphaera, Bacteroides, and Solobacterium, indicating strong microbial interactions that enhance caproate production. These findings suggest that producing MCCAs from expired dairy and beverage waste via lactate-mediated chain elongation is a viable method for sustainable waste management and could serve as a chemical production platform in the context of building a circular bioeconomy.

Quality comparison of non-thermal sterilized raw apple vinegar and commercial apple vinegar products

A nonthermally sterilized raw apple vinegar was manufactured using an ultra-fine filtration process (0.2 μm membrane filter) and its quality was comparable to commercially available vinegar products. First, using apple concentrate as a raw material, it was possible to produce non-thermal sterilized Using a two-stage fermentation process of alcohol and acetic acid fermentations, a non-thermally sterilized raw apple vinegar with pH 2.94 and an acidity of 6.20% was produced from an apple concentrate. The fermentation process increased the browning index significantly. However, the fundamental quality parameters of the non-thermal sterilized raw apple vinegar (A) with sterilized apple vinegar (B) did not differ significantly. The pH (2.92-2.95) of apple vinegar (A and B) was higher than that (pH 2.65-2.70) of commercial vinegar (C and D), and the total acidity, which is in the range of 6.20-6.21% and 6.53-6.90%, respectively, was higher in samples C and D than in samples A and B. However, four kinds of organic acids were detected in non-thermal sterilized raw apple vinegar (A), and its total organic acid content (6,245.00 mg%) was significantly higher than that of other samples (B, C, D) (p<0.05). In particular, malic acid content, as a main organic acid in apples, was very high in sample (A) (244.83 mg%) and sample (B) (210.21 mg%), compared to commercial products C (125.78 mg%) and D (86.90 mg%). The total polyphenol content and antioxidant activity of fermented apple vinegar (A, B) were more than twice as high as those of commercial products (C, D). Vinegar A had higher total polyphenol content than vinegar B. The above results suggest it is possible to manufacture and commercialize non-thermal sterilized raw apple vinegar with higher organic acid content and antioxidant properties using ultra-fine filtration.

Efficient production of R-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana using biofilm-based two-stage fermentation

In this work, a novel biofilm-based fermentation of Beauveria bassiana was employed to convert R-2- phenoxypropionic acid (R-PPA) to R-2-(4-hydroxyphenoxy) propionic acid (R-HPPA). The biofilm culture model of Beauveria bassiana produced a significantly higher R-HPPA titer than the traditional submerged fermentation method. Mannitol dosage, tryptone dosage, and initial pH were the factors that played a significant role in biofilm formation and R-HPPA synthesis. Under the optimal conditions, the maximum R-HPPA titer and productivity approached 22.2 g/L and 3.2 g/(L·d), respectively. A two-stage bioreactor combining agitation and static incubation was developed to further increase R-HPPA production. The process was optimized to achieve 100 % conversion of R-PPA, with a maximum R-HPPA titer of 50 g/L and productivity of 3.8 g/(L·d). This newly developed biofilm-based two-stage fermentation process provides a promising strategy for the industrial production of R-HPPA and related hydroxylated aromatic compounds.

Evaluation of safety and quality parameters of persimmon balsamic-type vinegar during multistarter culture fermentation and accelerated aging with oak chips

The present study explores for the first time, the feasibility of using concentrated persimmon juice (CPJ) as a starting material for high-quality balsamic-type vinegar (cv. Jiro, Northern Greece) via a two-stage fermentation process (alcoholic fermentation and then acetification). The best-performing wild type osmophilic strains of Hanseniaspora uvarum and Saccharomyces cerevisiae and acetic acid bacteria isolated from concentrated grape must and varietal vinegar (cv. Xinomavro, Northen Greece), respectively, were used as multistarters. The objective was to produce persimmon balsamic vinegar (PBV) that could be subsequently aged using oak chips as an accelerating agent under conditions that promote its quality attributes. A Taguchi design was applied to reveal the most influential combination of process factors (oak chips amount and pretreatment, aging time) as to the desired sensorial and physicochemical characteristics of the aged PBV (saturated and clear red-orange color, balanced ratio of sugars/organic acids, low 5-hydroxymethyl furfural (HMF) content, high antioxidant activity). Notably, adding even very low amounts (1% w/v) of oak chips significantly affected the quality profile of PBV within a very short aging time (10 days). After 30 days, PBV presented distinct, attractive color characteristics regardless of the amount or pretreatment of oak chips. The HMF content remained constantly low during all treatments while the redox potential increased on intensification of conditions. Implications for the role of phenolic compounds and/or other component interactions with oak chips are discussed. Our approach and outcomes contribute to the innovative valorization of persimmon fruits and the development of aged persimmon balsamic vinegar.

Synergistic approach for enhanced production of polyhydroxybutyrate by Bacillus pseudomycoides SAS-B1: Effective utilization of glycerol and acrylic acid through fed-batch fermentation and its environmental impact assessment

The continual rise in the consumption of petroleum-based synthetic polymers raised a significant environmental concern. Bacillus pseudomycoides SAS-B1 is a gram-positive rod-shaped halophilic bacterium capable of accumulating Polyhydroxybutyrate (PHB)-an intracellular biodegradable polymer. In the present study, the optimal conditions for cell cultivation in the seed media were developed. The optimal factors included a preservation age of 14 to 21 days (with 105 to 106 cells/mL), inoculum size of 0.1 % (w/v), 1 % (w/v) glucose, and growth temperature of 30 °C. The cells were then cultivated in a two-stage fermentation process utilizing glycerol and Corn Steep Liquor (CSL) as carbon and nitrogen sources, respectively. PHB yield was effectively increased from 2.01 to 9.21 g/L through intermittent feeding of glycerol and CSL, along with acrylic acid. FTIR, TGA, DSC, and XRD characterization studies were employed to enumerate the recovered PHB and determine its physicochemical properties. Additionally, the study assessed the cradle-to-gate Life Cycle Assessment (LCA) of PHB production, considering net CO2 generation and covering all major environmental impact categories. The production of 1000 kg of PHB resulted in lower stratospheric ozone depletion and comparatively reduced carbon dioxide emissions (2022.7 kg CO2 eq.) and terrestrial ecotoxicity (9.54 kg 1,4-DCB eq.) than typical petrochemical polymers.

Engineering photo-methylotrophic Methylobacterium for enhanced 3-hydroxypropionic acid production during non-growth stage fermentation

This study explored the potential of methanol as a sustainable feedstock for biomanufacturing, focusing on Methylobacterium extorquens, a well-established representative of methylotrophic cell factories. Despite this bacterium’s long history, its untapped photosynthetic capabilities for production enhancement have remained unreported. Using genome-scale flux balance analysis, it was hypothesized that introducing photon fluxes could boost the yield of 3-hydroxypropionic acid (3-HP), an energy- and reducing equivalent-consuming chemicals. To realize this, M. extorquens was genetically modified by eliminating the negative regulator of photosynthesis, leading to improved ATP levels and metabolic activity in non-growth cells during a two-stage fermentation process. This modification resulted in a remarkable 3.0-fold increase in 3-HP titer and a 2.1-fold increase in its yield during stage (II). Transcriptomics revealed that enhanced light-driven methanol oxidation, NADH transhydrogenation, ATP generation, and fatty acid degradation were key factors. This development of photo-methylotrophy as a platform technology introduced novel opportunities for future production enhancements.

Enhanced production of biohydrogen and biomethane through a two-stage anaerobic fermentation of food waste mixed with conductive additives

This study was conducted to examine the effects of three different conductive materials (CMs) on H2 and CH4 production in a two-stage fermentation process (TSFP). This study also investigated the distinct roles played by the different types of CMs within the process. The study findings revealed that the combination of two or more CMs led to a significant improvement in production compared to using a single CM. Magnetite nanoparticle (MNP) played a crucial role in enhancing H2 productivity during the first stage of the TSFP. The TSFP with carbon nanotubes (CNT) + MNP demonstrated the highest H2 yield of 121 ± 7 mL/g·COD with the H2 production rate of 219 ± 18 mL/hr, followed by the TSFP with MNP alone yielded the second highest H2 of 81 ± 5 mL/g·COD with the H2 production rate of 169 ± 25 mL/hr. Both powdered activated carbon (PAC) alone (277 ± 17 mL/g·COD) and PAC + CNT (278 ± 15 mL/g·COD) showed the highest CH4 yield, indicating that PAC contributed to higher CH4 production in the second stage of the TSFP. The addition of PAC to the samples led to higher chemical oxygen demand (COD) removal efficiency in the TSFP, while the presence of MNP significantly increased the total energy production.

Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding

Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.

Time-resolved transcriptomic profile of oleaginous yeast Rhodotorula mucilaginosa during lipid and carotenoids accumulation on glycerol

The study reports the exploration of the transcriptome landscape of the red oleaginous yeast Rhodotorula mucilaginosa IIPL32 coinciding with the fermentation kinetics of the yeast cultivated in a two-stage fermentation process to exploit the time-series approach to get the complete transcripts picture and reveal the persuasive genes for fatty acid and terpenoid synthesis. The finding displayed the molecular drivers with more than 2-fold upregulation in the nitrogen-limited stage than in the nitrogen-excess stage. The rate-limiting diphosphomevalonate decarboxylase, acetylCoA-citrate lyase, and acetyl-CoA C-acetyltransferase were significant in controlling the metabolic flux in the synthesis of reduced compounds, and acetoacetyl-CoA synthase, 3-ketoacyl-acyl carrier-protein reductase, and β-subunit enoyl reductase catalyze the key starting steps of lipids or terpenoid synthesis. The last two catalyze essential reduction steps in fatty acid synthesis. These enzymes would be the prime targets for the metabolic engineering of the oleaginous yeast for enhanced fatty acids and terpenoid production.

Screening and Identification of the Strain Pediococcus acidilactici and Its Application in Fermentation of Corn–Soybean Meal Uncooked Materials

Cost and contamination are the bottleneck problems for the replacement of antibiotics with fermented feed. A strain of lactic acid bacteria was isolated and screened from acidified apple juice and identified as Pediococcus acidilactici using morphological, physiological, and biochemical tests and 16S rDNA sequence analysis. The strain was combined with Bacillus subtilis and Saccharomyces cerevisiae to ferment raw corn–soybean meal, and the two-stage fermentation process was optimized with a single-factor test, orthogonal test, and response surface methodology. Compared with unfermented raw material, the protein content of fermented feed was increased by 5.21 percentage points (p < 0.05) and the total amino acids were increased by 3.7 percentage points (p < 0.05), making it rich in amino acids essential for pigs. The high-throughput sequencing results showed that, at the species level, the highest relative abundances of bacteria in the fermentation system were those of Pediococcus acidilactici and Bacillus subtilis, and the most abundant fungi was Saccharomyces cerevisiae. No pathogenic bacteria, such as Salmonella, were detected in the fermentation system. This paper provides a feasible scheme for cheap preparation of an alternative to antibiotics, fermented feed, with uncooked raw materials. It has positive significance for promoting high-value utilization of agricultural and sideline products and improving feed cost-effectiveness.

Phenolic Biotransformations in Wheatgrass Juice after Primary and Secondary Fermentation.

Fermented wheatgrass juice was prepared using a two-stage fermentation process by employing Saccharomyces cerevisiae and recombinant Pediococcus acidilactici BD16 (alaD+). During fermentation, a reddish-brown hue appeared in wheatgrass juice due to production of different types of red pigments. The fermented wheatgrass juice has considerably higher content of anthocyanins, total phenols and beta-carotenes as compared to unfermented wheatgrass juice. It has low ethanol content, which might be ascribed to the presence of certain phytolignans in wheatgrass juice. Several yeast-mediated phenolic transformations (such as bioconversion of coumaric acid, hydroxybenzoic acid, hydroxycinnamic acid and quinic acid into respective derivatives; glycosylation and prenylation of flavonoids; glycosylation of lignans; sulphonation of phenols; synthesis of carotenoids, diarylnonanoids, flavanones, stilbenes, steroids, quinolones, di- and tri-terpenoids and tannin) were identified in fermented wheatgrass juice using an untargeted liquid chromatography (LC)-mass spectrometry (MS)-matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/time-of-flight (TOF) technique. The recombinant P. acidilactici BD16 (alaD+) also supported flavonoid and lignin glycosylation; benzoic acid, hydroxycoumaric acid and quinic acid derivatization; and synthesis of anthraquinones, sterols and triterpenes with therapeutic benefits. The information presented in this manuscript may be utilized to elucidate the importance of Saccharomyces cerevisiae and P. acidilactici BD16 (alaD+) mediated phenolic biotransformations in developing functional food supplements such as fermented wheatgrass juice.

Two-stage fermentation enhanced single-cell protein production by Yarrowia lipolytica from food waste

The resource utilization of food waste is crucial, and single-cell protein (SCP) is attracting much attention due to its high value. This study aimed to convert food waste to SCP by Yarrowia lipolytica. It was found the chemical oxygen demand (COD) removal rate 77 ± 1.70% was achieved at 30 g COD/L with the protein content of biomass only 24.1 ± 0.4% w/w biomass dry weight (BDW) in one-stage fermentation system. However, the protein content was significantly increased to 38.8 ± 0.2% w/w BDW with the COD removal rate 85.5 ± 0.7% by a two-stage fermentation process, where the food waste was firstly anaerobically fermented to volatile fatty acids and then converted to SCP with Yarrowia lipolytica. Transcriptomic analysis showed that the expression of SCP-producing genes including ATP citrate (pro-S)-lyase and fumarate hydratase class II were up-regulated in the two-stage transformation, resulting in more organic degradation for SCP synthesis.

Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

Clonostachys rosea: Production by Submerged Culture and Bioactivity Against Sclerotinia sclerotiorum and Bemisia tabaci.

Among the prospective biocontrol agents, the saprophytic filamentous fungus Clonostachys rosea is an excellent necrotrophic mycoparasite of numerous plant pathogenic fungi. However, its commercial development has been hampered by mass production difficulties during solid-state fermentation. Conversely, the submerged liquid fermentation shortens the cultivation time while increasing yields of fungal propagules. However, this method has been overlooked for C. rosea. In this work, we investigated the impact of liquid pre-culture inoculum on the spore production by the two-stage fermentation process using rice grains in comparison to the traditional solid-state fermentation. In parallel, we studied the submerged cultivation of C. rosea by manipulating carbon-to-nitrogen (C:N) ratio and nitrogen source, with the further optimization of spore production in a benchtop bioreactor. Additional bioassays included assessing the bioactivity of water-dispersible microgranules (that contained a submerged conidia) against the whitefly (Bemisia tabaci biotype B) and Sclerotinia sclerotiorum (causal agent of the white mold). Our results showed a maximum concentration of 1.1 × 109 conidia/g-dry-matter after 7 days of cultivation by two-stage fermentation process. The liquid fermentation yielded 1.4 × 109 submerged conidia/ml after 7 days using a medium with a 50:1 C:N ratio, and it also induced the production of microsclerotia (MS) up to 1.35 × 104/ml within 6 days with 10:1 C:N ratio; both media were supplemented with dextrose monohydrate and soybean meal. The fermentation batches carried out in a benchtop bioreactor with medium 50:1 C:N ratio and amended with soybean meal rendered a production peak on the fourth day, corresponding to 1.11 × 109 conidia/ml and 4.35 × 108 colony forming units (CFU)/ml. Following air-drying, the conidia production from air-dried microgranules of C. rosea biomass was estimated at 3.4 × 1010 conidia/g of formulated product upon re-hydration for 7 days. Both submerged conidia and MS of C. rosea inhibited 100% germination of S. sclerotiorum sclerotia by direct parasitism. The air-dried submerged conidia exhibited a suppressive activity on sclerotia (88% mycoparasitism) and early whitefly nymphs (76.2% mortality) that rendered LC50 values of 3.2 × 104 CFU/g soil and 1.5 × 107 CFU/ml, respectively. Therefore, the submerged liquid culture of C. rosea may offer a feasible and cost-effective method for its large-scale production, alleviating critical constraints to their commercial use while providing an additional tool for management of B. tabaci and S. sclerotiorum.

Co-production of Biohydrogen and Biomethane from Chicken Manure and Food Waste in a Two-Stage Anaerobic Fermentation Process.

Batch experiments were performed to evaluate the biohydrogen and biomethane production by co-digestion of chicken manure and food waste in a two-stage mesophilic fermentation process. Results showed that no hydrogen was produced in the first stage of sole chicken manure fermentation, while methane yield was 247.3 mL·g-1-VS. By comparison, the co-digestion process with food waste proportions of 50-85% obtained hydrogen yields of 15.5-57.5 mL·g-1-VS, and the methane yields and maximum specific methane production rates were also improved by 7.0-16.7% and 80%, respectively. Moreover, the highest hydrogen and methane yields were achieved during sole food waste fermentation process. The acetate was the main volatile fatty acid (VFA) produced during sole chicken manure fermentation process in the first stage. Statistical analysis revealed that hydrogen production from co-digestion process and sole food waste fermentation process followed the n-butyrate-type pathway. Meanwhile, it should be noticed that the co-fermentation of chicken manure and food waste had antagonistic effects on the hydrogen fermentation, implying that there might be some inhibition factors existing in chicken manure or produced during the co-fermentation process. At the beginning of methane fermentation, the VFA profiles were similar to those at the end of hydrogen fermentation, and the main VFA compositions changed to acetate and propionate in the latter period of methane production. The volatile solid removal efficiencies were also promoted in co-digestion process compared with sole chicken manure digestion, which were increased by 9.7-14.4% with food waste proportions of 50-80%.

Effective utilization of refractory dissolved organic matters in domestic sewage allows to enhanced nitrogen removal by integrated fermentation, nitrification, denitratation and anammox process

To take full advantage of refractory dissolved organic matters (rDOMs) and generate sufficient nitrate for domestic sewage treatment, this study presented a novel integrated fermentation, nitrification, denitratation and anammox (IFNDA) process in a combined ABR-CSTR reactor. The results showed that an advanced total nitrogen (TN) removal efficiency of 94.1% was obtained after over 190 days operation, resulting in effluent TN concentration as low as 3.6 mg/L. The system nitrogen removal was dominated by anammox with a high proportion of 88.6%. The high conversion rate of acetic acid (54.0%) and volatile fatty acids (64.5%) from rDOMs in domestic sewage by in-situ fermentation drove efficient denitratation. Microflora analysis indicated that the enriched Commamonas (3.5%) and Longilinea (3.3%) dominated hydrolysis and acidogenesis of organics, and Methanosaeta (9.0%) obligated acetoclastic methanogenesis in two-stage fermentation process. Thauera (8.4%) and Candidatus Brocadia (2.5%) were the core bacteria for nitrogen metabolism in the IFNDA system.

Functional characterization of a novel violacein biosynthesis operon from Janthinobacterium sp. B9-8.

Violacein is a secondary metabolite mainly produced by Gram-negative bacteria that is formed from tryptophan by five enzymes encoded by a single operon. It is a broad-spectrum antibacterial pigment with various important biological activities such as anti-tumor, antiviral, and antioxidative effects. The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8. The key enzyme-encoding genes were cloned to construct the multigene coexpression plasmids pET-vioAB and pRSF-vioCDE. The violacein biosynthesis pathway was heterologously introduced into engineered Escherichia coli VioABCDE and VioABCDE-SD. The factors affecting violacein production, including temperature, pH, inoculum size, carbon and nitrogen source, precursor, and inducers were investigated. The violacein titer of VioABCDE-SD reached 107mg/L in a two-stage fermentation process, representing a 454.4% increase over the original strain. The violacein operon from B9-8 provides a new microbial gene source for the analysis of the violacein synthesis mechanism, and the constructed engineering E. coli strains lay a foundation for the efficient and rapid synthesis of other natural products.Key points• The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8.• The violacein synthesis pathway was reconstructed in E. coli using two compatible plasmids.• A two-stage fermentation process was optimized for improved violacein accumulation.

Optimization and feedback control system of dilution rate for 1,3-propanediol in two-stage fermentation: A theoretical study.

In utilizing glycerol to produce 1,3-propanediol by microbial fermentation, the problems of low utilization rate and poor production performance need to be addressed. Based on the analysis of a mathematical model for 1,3-propanediol production from glycerol by Klebsiella pneumoniae, this study theoretically investigated the effects of the dilution rate and the initial glycerol concentration in a two-stage fermentation process and the feasibility of applying the feedback control methods. First, the optimal operation conditions of initial glycerol concentration and dilution rate were obtained. Through the use of feedback control theory, a control strategy for dilution rate was designed and optimized to shorten the settling time (time required for fermentation to reach stability) from 60.92 to 36.68 h for the first reactor, and from 53.66 to 22.68 h for the second reactor. In addition, the yield of 1,3-propanediol in both two reactors reached up to 0.5g·g-1 . The simulation results indicated that the feedback control strategy for dilution rate increased the product concentration, reduced the residual glycerol in the fermentation broth, and greatly improved the performance of the fermentation. A feeding strategy of automatic control for dilution rate has been established and will be applied as an effective guiding scheme in automatic continuous fermentations for production of 1,3-propanediol.

Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.