Two-step Fermentation Process Research Articles

Chemical profiling and probiotic viability assessment in Gueuze-style beer: Fermentation dynamics, metabolite and sensory characterization, and in vitro digestion resistance

Probiotic viability, metabolite concentrations, physicochemical parameters, and volatile compounds were characterized in Gueuze beers formulated with probiotic lactic acid bacteria (LAB) and yeast. Additionally, the sensory profile of the beers and the resistance of the probiotics to digestion were determined. The use of 2 International Bitterness Units resulted in high concentrations of probiotic LAB but a decline in probiotic yeast as pH decreased. Secondary fermentation led to the consumption of maltose, citric acid, and malic acid, and the production of lactic and propionic acids. Carbonation and storage at 4 °C had minimal impact on probiotic viability. The addition of probiotic LAB resulted in a distinct aroma profile with improved sensory characteristics. Our results demonstrate that sour beers produced with probiotic LAB and a probiotic yeast, and fermented using a two-step fermentation process, exhibited optimal physicochemical parameters, discriminant volatile compound profiles, promising sensory characteristics, and high probiotic concentrations after digestion.

Scalable Ammonia Synthesis in Fermentors Using Quantum Dot-Azotobacter vinelandii Hybrids

AbstractThis study introduces a scalable synthesis of ammonia through photochemical reactions, wherein nitrogen-fixing bacterial cells, Azotobacter vinelandii (A. vinelandii), form hybrids with colloidal quantum dots (QDs). Irradiation of the QD-A. vinelandii hybrids with visible light is found to significantly enhance ammonia production efficiency. The inherently low ammonia conversion rate of wild-type A. vinelandii is substantially increased upon incorporation of QDs. This increase is attributed to the electron transfer from QDs within the bacterial cells to intracellular bio-components. Transferring this chemistry to a large-scale reaction presents a tremendous challenge, as it requires precise control over the growth conditions. We explore the scalability of the QD-A. vinelandii hybrids by conducting the photochemical reaction in a 5-L fermentor under various parameters, such as dissolved oxygen, nutrient supply, and pH. Interestingly, ammonia was produced in media depleted of carbon sources. Consequently, a two-step fermentation process was designed, enabling effective ammonia production. Our findings demonstrate that the QD-A. vinelandii hybrid system in a bioreactor setup achieves an ammonia turnover frequency of 11.96 s−1, marking a more than sixfold increase in efficiency over that of nitrogenase enzymes alone. This advancement highlights the potential of integrating biological and nanotechnological elements for scalable ammonia production processes.

The Chemical Profiles and Antioxidant Properties of Live Fruit or Vegetable Vinegars Available on the Polish Food Market.

Live vinegar is a product formed through a two-step fermentation process of a sugar substrate that has not been subjected to filtration or pasteurization. This is considered to preserve all nutrients and biologically active microorganisms, making it a product with a valuable composition and beneficial properties. Therefore, the purpose of this study was to analyze the chemical composition and antioxidant properties of the selected vinegars available on the Polish food market. The material in the study consisted of four live (naturally turbid, unfiltered, unpasteurized) fruit or vegetable vinegars: apple, pear, rhubarb, and lemon. Spectrophotometric, HPLC, and GC methods were used. Among the vinegars tested, lemon vinegar had the highest vitamin C content-15.95 mg/100 mL. Apple vinegar proved to be the best source of polyphenols and flavonoids (TPC-191.97 mg GAE/L, TFC-70.22 mg RE/L). All of the vinegars contained dihydroxybenzoic acid, 4-hydroxybenzoic acid, caffeic acid, 2-hydroxycinnamic acid, and myricetin. The acetic acid content of the tested vinegars ranged from 29.180 to 38.125 mM/L. The pH values ranged from 3.14 to 3.41. In conclusion, the most promising nutraceutical with potentially beneficial health-promoting properties seems to be apple vinegar.

Recent Advances in 2-Keto-l-gulonic Acid Production Using Mixed-Culture Fermentation and Future Prospects.

Vitamin C, also known as ascorbic acid, is an essential vitamin that cannot be synthesized by the human body and must be acquired through our diet. At present, the precursor of vitamin C, 2-keto-l-gulonic acid (2-KGA), is typically produced via a two-step fermentation process utilizing three bacterial strains. The second step of this traditional two-step fermentation method involves mixed-culture fermentation employing 2-KGA-producing bacteria (Ketogulonicigenium vulgare) along with associated bacteria. Because K. vulgare has defects in various metabolic pathways, associated bacteria are needed to provide key substances to promote K. vulgare growth and 2-KGA production. Unlike previous reviews where the main focus was the interaction between associated bacteria and K. vulgare, this Review presents the latest scientific research from the perspective of the metabolic pathways associated with 2-KGA production by K. vulgare and the mechanism underlying the interaction between K. vulgare and the associated bacteria. In addition, the dehydrogenases that are responsible for 2-KGA production, the 2-KGA synthesis pathway, strategies for simplifying 2-KGA production via a one-step fermentation route, and, finally, future prospects and research goals in vitamin C production are also presented.

Microbial Interactions in a Vitamin C Industrial Fermentation System: Novel Insights and Perspectives.

In industrial production, the precursor of l-ascorbic acid (L-AA, also referred to as vitamin C), 2-keto-l-gulonic acid (2-KLG), is mainly produced using a classic two-step fermentation process performed by Gluconobacter oxydans, Bacillus megaterium, and Ketogulonicigenium vulgare. In the second step of the two-step fermentation process, the microbial consortium of K. vulgare and B. megaterium is used to achieve 2-KLG production. K. vulgare can transform l-sorbose to 2-KLG, but the yield of 2-KLG is much lower in the monoculture than in the coculture fermentation system. The relationship between the two strains is too diverse to analyze and has been a hot topic in the field of vitamin C fermentation. With the development of omics technology, the relationships between the two strains are well explained; nevertheless, the cell-cell communication is unclear. In this review, based on current omics results, the interactions between the two strains are summarized, and the potential cell-cell communications between the two strains are discussed, which will shed a light on the further understanding of synthetic consortia.

Cost-effective desulfurization of acid mine drainage with food waste as an external carbon source: A pilot-scale and long-term study

Sulfate reduction process can be a promising method for simultaneously removing sulfate , metals and metalloids from acid mine drainage (AMD). However, organic matter in AMD is far from enough for sulfate reduction, and an additional carbon source is required, which increases operation costs for AMD treatment. In this study, a two stage AMD treatment system was established (chemical precipitation - sulfate reduction), and food waste hydrolysate was utilized as a carbon source for AMD treatment. Simultaneous removal of sulfate, organics, metals and metalloids was observed in a pilot-scale (10 m 3 ) upflow anaerobic sludge bed (UASB) for over 400 days. Introducing of food waste hydrolysate (FWHS) was not harmful for sulfate reducers in UASB. Decomposition of refractory organic matter in AMD was enhanced after FWHS addition, which led to decreasing of COD concentration (50%) in effluents of UASB. Ten out of 17 organic matter in effluents was no longer detected after FWHS addition. Metals and metalloids from both AMD and FWHS were efficiently removed during the sulfate reduction process (97.7–100%). Volatile fatty acids (acetic and valeric acids) produced during the food waste hydrolysis could be utilized as carbon source for AMD treatment. Interactions among fermentative microbes , sulfate reducers, denitrifiers and methanogens enhanced carbon, sulfur and nitrogen removal in the UASB. Therefore, a cost-effective method for AMD and food waste cotreatment (3.6 kgSO 4 2− /kgTS food waste) was established in this study by reducing carbon source addition (100%), biomass production (93%), VFA production (54.5%) and sulfide emission (50%), which might be applied for AMD recycling at mining sites. • a two-step fermentation and sulfate reduction coupling process was established. • Food waste was used as carbon and nutrients source for acid mine drainage treatment. • Sulfate, carbon, metals and metalloids were efficiently removed (87.3–100%). • The established technology might be used for acid mine drainage treatment in mines.

Recycling Food Waste and Saving Water: Optimization of the Fermentation Processes from Cheese Whey Permeate to Yeast Oil

With the aim of developing bioprocesses for waste valorization and a reduced water footprint, we optimized a two-step fermentation process that employs the oleaginous yeast Cutaneotrichosporon oleaginosus for the production of oil from liquid cheese whey permeate. For the first step, the addition of urea as a cost-effective nitrogen source allowed an increase in yeast biomass production. In the second step, a syrup from candied fruit processing, another food waste supplied as carbon feeding, triggered lipid accumulation. Consequently, yeast lipids were produced at a final concentration and productivity of 38 g/L and 0.57 g/L/h respectively, which are among the highest reported values. Through this strategy, based on the valorization of liquid food wastes (WP and mango syrup) and by recovering not only nutritional compounds but also the water necessary for yeast growth and lipid production, we addressed one of the main goals of the circular economy. In addition, we set up an accurate and fast-flow cytometer method to quantify the lipid content, avoiding the extraction step and the use of solvents. This can represent an analytical improvement to screening lipids in different yeast strains and to monitoring the process at the single-cell level.

Integrated production of ethanol and xylitol from Brassica juncea using Candida sojae JCM 1644

The present study demonstrates a novel strategy involving two-step fermentation of lignocellulosic hydrolysate for the integrated production of ethanol and xylitol using a newly isolated yeast strain, Candida sojae JCM 1644. The isolated strain was characterised by its carbohydrate assimilation efficiency and tolerance towards inhibitors generated during pretreatment and fermentation of lignocellulosic biomass. In brief, the study comprised alkali treatment of Brassica juncea followed by its saccharification with cellulase consortia. An isolated strain was used for the co-production of xylitol and ethanol from sugar hydrolysate, and several parameters were systematically optimised for maximum co-production of ethanol and xylitol. Out of total glucose (149.72 g/L) and xylose (84.21 g/L) present in biomass hydrolysate, a product yield of 0.45 g/g (ethanol) and 0.62 g/g (xylitol) was achieved for a two-step fermentation process, which was 15.57% and 11.78% higher than the yield achieved for ethanol and xylitol, respectively, in a one-step fermentation process.

Applications of one-Step environmentally-friendly fermentation method to produce fumaric acid with immobilized Rhizopus arrhizus in stirred-tank reactor

The traditional two-step cultivation mode for fungal cultivation is commonly divided into the seed stage (usually 24 to 48 h) and the production stage (usually 96 to 144 h). The use of two stages prolongs the total production cycle and generates excess wastewater. In this work, an efficient and environmentally friendly one-step fermentation method was applied for the production of fumaric acid by immobilized Rhizopus arrhizus in a stirred-tank reactor. The nitrogen source content and the agitation speed of the reactor were optimized as the two critical factors in the one-step fermentation process; the fumaric acid production of 51 g/L with a yield of 0.42 g/g glucose was comparable with the product from the traditional two-step fermentation process. Furthermore, the total production cycle was shortened to 96 h, and the amount of wastewater was reduced due to the avoidance of the seed culture process. Thus, utilization of the one-step fermentation method to produce fumaric acid was shown to be preferable feasibility and environmental-friendliness. This is a promising method for industrial production of fumaric acid and other high-value biochemicals by fungus.

Techno-economic analysis of a two-step fermentation process for bio-butanol production from cooked rice

Biobutanol was produced from cooked rice by utilizing two steps: microbial saccharification followed by aerobic acetone–butanol–ethanol (ABE) fermentation. The scaled-up process was simulated via SuperPro Designer®.

Coproduction of hydrogen and volatile fatty acids via integrated two-step fermentation of sweet sorghum stalks by alkaline and enzymatic treatment

Coproduction of H2 and volatile fatty acids from sweet sorghum stalks was successfully developed via two-step fermentation process involving alkaline and enzymatic treatment of the residual slurry obtained from the first step fermentation of the raw material. The optimum treatment conditions to obtain the highest yield of the end products included 2% (w/v) alkali at 120 °C for the residual slurry and 32 FPU cellulase per gram of the alkali-treated materials. The two-step fermentation using Clostridium thermosaccharolyticum increased the end product yields of H2 (6.37 mmol/g-substrate) by 95%, acetic acid (2.33 g/L) by 97% and butyric acid (2.36 g/L) by 143% compared with those obtained in the single step fermentation without any treatment. As a result, the H2 energy recovery efficiency of the two-step fermentation process with commercially attractive butyric acid as a coproduct reached 10.54%. These results provide a promising approach for sweet sorghum biorefining.

Transcriptome Analysis of Gluconobacter oxydans WSH-003 Exposed to Elevated 2-Keto-L-Gulonic Acid Reveals the Responses to Osmotic and Oxidative Stress.

Industrial production of 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C, is mainly achieved by a two-step fermentation process carried out by Gluconobacter oxydans, Bacillus, and Ketogulonicigenium. One of the most promising innovations that could replace this complicated two-step fermentation process is the integration of the essential genes for synthesis of 2-KLG into G. oxydans and use of it as the producer. Therefore, determining the tolerance and response of G. oxydans to 2-KLG is a priority for improving the direct production of 2-KLG in this bacterium. In this study, a global view of the gene expression of G. oxydans WSH-003 in response to 2-KLG challenge was investigated by RNA sequencing. A total of 363 genes of G. oxydans that were differentially expressed in response to 2-KLG were uncovered. The results showed that 2-KLG could lead to oxidative stress, osmotic stress, and DNA damage in G. oxydans.

Efficient Optimization ofGluconobacter oxydansBased on Protein Scaffold-Trimeric CutA to Enhance the Chemical Structure Stability of Enzymes for the Direct Production of 2-Keto-L-gulonic Acid

2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is produced by a two-step fermentation route from D-sorbitol in industry. However, this route is a complicated mix-culture system which involves three bacteria. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. The one-step fermentation of 2-keto-L-gulonic acid (2-KLG) has been achieved in our previous study; 32.4 g/L of 2-KLG production was obtained by the one-step strainG. oxydans/pGUC-tufB-sdh-GGGGS-sndhafter 168 h. In this study, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) were expressed inG. oxydansafter the codon optimization. Furthermore, the trimeric protein CutA was used to improve the chemical structure stability of SDH and SNDH. The recombinant strainG. oxydans/pGUC-tufB-SH3-sdh-GGGGS-sndh-tufB-SH3lig-(GGGGS)2-cutAproduced 40.3 g/L of 2-KLG after 168 h. In addition, the expression levels of the cofactor PQQ were enhanced to further improve 2-KLG production. With the stepwise metabolic engineering ofG. oxydans, the final 2-KLG production was improved to 42.6 g/L. The efficient one-step production of 2-KLG was achieved, and the final one-step industrial-scale production of 2-KLG is drawing near.

Engineering bacteria-seaweed symbioses for modulating the photosynthate content of Ulva (Chlorophyta): Significant for the feedstock of bioethanol production

Seaweed biomass cultivation predicates the quantity and quality of this biorefinery feedstock. Unfortunately, the seaweed growth rate and chemical content are hardly predictable and are affected by environmental factors, including epiphytic bacteria. We hypothesize that microbiome engineering can control the chemical composition of Ulva biomass. We show that the engineered Maribacter sp. and Roseovarius sp. consortium modulate Ulva mutabilis growth rate and photosynthate content of constituents relevant for bioethanol production. Although minimal growth was observed in the axenic cultures (0.04 mm day−1), Ulva mutabilis in a tripartite community showed a growth rate of 3.79 mm day−1 in the growth phase. Furthermore, the content of glucose and glycerol in Ulva of the engineered community increased by 77 ± 19% and 460 ± 207% whereas xylose and glucuronic acid decreased by 37 ± 14% and 46 ± 15% in comparison to axenic culture.Interestingly, bacterial addition affected the rhamnose/xylose/glucuronic acid ratio (1.96:1:1: vs 1.34:0.85:1 in xenic vs axenic culture), indicating the impact of bacteria on ulvan synthesis. In addition, tyrosine and histidine increased by 191 ± 61% and 40 ± 26%; however, valine, isoleucine, aspartate, threonine, serine, and phenylalanine decreased by 22 ± 19% - 42 ± 23%. Flux-balance analysis of Saccharomyces cerevisiae, Escherichia coli, and Clostridium acetobutylicum was used to estimate the bioethanol yield from hydrolyzed Ulva biomass, in a one-step or two-step fermentation process. Simulation using S. cerevisiae (RN1016) with xylose isomerase resulted in a bioethanol yield of 85.62 for xenic vs. 71.31 mg/g dry weight (DW) axenic cultures of Ulva.The increased growth rate and the relative amounts of photosynthates of U. mutabilis are modulated by the engineered microbiome. Moreover, it results in biomass with a higher potential for bioethanol fermentation in comparison to axenic cultures.

High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase.

2-Keto-L-gulonic acid (2-KLG) is the direct precursor for the production of L-ascorbic acid (L-Asc) on industrial scale. Currently, the production of L-Asc in the industry is a two-step fermentation process. Owing to many unstable factors in the fermentation process, the conversion rate of L-sorbose to 2-KLG has remained at about 90% for many years. In order to further improve the production efficiency of 2-KLG, a FAD-dependent sorbose dehydrogenase (SDH) has been obtained in our previous research. The SDH can directly convert L-sorbose to 2-KLG at a very high efficiency. However, the enzyme activity of the SDH is relatively low. In order to further improve the enzyme activity of the SDH, a high throughput screening platform the dehydrogenase is essential. By optimizing the promoter, host and sorbosone dehydrogenase (SNDH), knockout of the aldosterone reductases and PTS related genes, a reliable platform for high-throughput screening of more efficient FAD-dependent SDH has been established. By using the high-throughput screening platform, the titer of the 2-KLG has been improved by 14.1%. The method established here could be useful for further enhancing the FAD-dependent SDH, which is important to achieve the efficient one-strain-single-step fermentation production of 2-KLG.

Biorefining Process of Carbohydrate Feedstock (Agricultural Onion Waste) to Acetic Acid

The biorefining of agricultural waste into green chemicals has clear potential for improving global environmental sustainability. In this study, we evaluated the potential of acetic acid production from carbohydrate feedstock (onion waste, OW) as a more environmentally friendly source than feedstock produced from natural gas. In particular, OW is an ideal feedstock for the biorefining process as it contains a sufficient amount of carbohydrates (69.7%). Five days of the simultaneous saccharification and two-step fermentation (SSTF) process produced acetic acid from OW more efficiently than the simultaneous saccharification and cofermentation (SSCF) process. SSTF produced 19.3 g/L acetic acid and recorded the highest conversion yield (90.5%) from OW (6% substrate loading, w/v). These results suggested that acetic acid can be efficiently and sustainably produced from OW by the SSTF process.

Evaluation of the Two-Stage Fermentative Hydrogen Production from Sugar Beet Molasses

Fermentative hydrogen production from molasses—a renewable by-product of beet-sugar processing—was considered. Technical and economic evaluations were performed of a stand-alone production plant employing a two-step fermentation process (dark thermophilic fermentation and photofermentation) followed by an adsorption-based upgrading of the produced hydrogen gas. Using a state-of-the-art knowledge base and a mathematical model composed of mass and energy balances, as well as economic relationships, the process was simulated and equipment data were estimated, the hydrogen cost was calculated and a sensibility analysis was carried out. Due to high capital, operating and labor costs, hydrogen production cost was estimated at a rather high level of 32.68 EUR/kg, while the energy output in produced hydrogen was determined as 68% more than the combined input of the thermal and electric energy needed for plant operation. As the room for improvement of plant performance is limited, a perspective on the cost competitiveness of large-scale hydrogen production from fossil sources is unclear.

Systematic characterization of sorbose/sorbosone dehydrogenases and sorbosone dehydrogenases from Ketogulonicigenium vulgare WSH-001

2-Keto-L-gulonic acid (2-KLG) is the direct precursor of vitamin C in industrial synthesis. 2-KLG is mainly produced via the classical two-step fermentation route. In the two-step fermentation process, 2-KLG can be synthesized from L-sorbose by Ketogulonicigenium vulgare aided by Bacillus megaterium. There are five sorbose/sorbosone dehydrogenases (SSDHs), SSDA1, SSDA1-P, SSDA2, SSDA3 and SSDB, and two sorbosone dehydrogenases (SNDHs), glucose/sorbosone dehydrogenase (GSNDH) and sorbosone dehydrogenase (SNDH), in K. vulgare, which could play crucial roles in transforming L-sorbose or L-sorbosone to 2-KLG. However, confusion about the catalytic characteristics of the individual SSDHs and SNDHs makes construction of a recombinational strain for the purpose of enhancing 2-KLG production difficult. In this study, the five SSDHs and two SNDHs from K. vulgare WSH-001 were purified, and their optimal pH values and reaction temperatures, kinetic properties, thermostabilities, substrate spectra and effects of electron acceptors on their performances were systematically determined. Among these dehydrogenases, only SSDA1 and SSDA3 have high activity for catalyzing L-sorbose to 2-KLG directly. These data provide more clues for ways to achieve enhanced conversion of L-sorbose in K. vulgare, which could facilitate both the construction of a more efficient one-step fermentation 2-KLG producer and the reconstruction of a one-step fermentation process.

Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in Gluconobacter oxydans.

L-erythrose, a rare aldotetrose, possesses various pharmacological activities. However, efficient L-erythrose production is challenging. Currently, L-erythrose is produced by a two-step fermentation process from erythritol. Here, we describe a novel strategy for the production of L-erythrose in Gluconobacter oxydans (G. oxydans) by localizing the assembly of L-ribose isomerase (L-RI) to membrane-bound sorbitol dehydrogenase (SDH) via the protein-peptide interactions of the PDZ domain and PDZ ligand. To demonstrate this self-assembly, green fluorescent protein (GFP) replaced L-RI and its movement to membrane-bound SDH was observed by fluorescence microscopy. The final L-erythrose production was improved to 23.5 g/L with the stepwise metabolic engineering of G. oxydans, which was 1.4-fold higher than that obtained using coexpression of SDH and L-RI in G. oxydans. This self-assembly strategy shows remarkable potential for further improvement of L-erythrose production.

Optimization of fermentation conditions for the production of curcumin by engineered Escherichia coli.

Curcumin is a plant secondary metabolite with outstanding therapeutic effects. Therefore, there is a great interest in developing new strategies to produce this high-value compound in a cheaper and environmentally friendly way. Curcumin heterologous production in Escherichia coli using artificial biosynthetic pathways was previously demonstrated using synthetic biology approaches. However, the culturing conditions to produce this compound were not optimized and so far only a two-step fermentation process involving the exchange of culture medium allowed high concentrations of curcumin to be obtained, which limits its production at an industrial scale. In this study, the culturing conditions to produce curcumin were evaluated and optimized. In addition, it was concluded that E. coli BL21 allows higher concentrations of curcumin to be produced than E. coli K-12 strains. Different isopropyl β-d-thiogalactopyranoside concentrations, time of protein expression induction and substrate type and concentration were also evaluated. The highest curcumin production obtained was 959.3 µM (95.93% of per cent yield), which was 3.1-fold higher than the highest concentration previously reported. This concentration was obtained using a two-stage fermentation with lysogeny broth (LB) and M9. Moreover, terrific broth was also demonstrated to be a very interesting alternative medium to produce curcumin because it also led to high concentrations (817.7 µM). The use of this single fermentation medium represents an advantage at industrial scale and, although the final production is lower than that obtained with the LB-M9 combination, it leads to a significantly higher production of curcumin in the first 24 h of fermentation. This study allowed obtaining the highest concentrations of curcumin reported so far in a heterologous organism and is of interest for all of those working with the heterologous production of curcuminoids, other complex polyphenolic compounds or plant secondary metabolites.