Two-stage Fermentation Research Articles

Biorefining process of agricultural onions to functional vinegar

Biorefinery of onion vinegar (OV) is attractive as a method for producing functional foods from onions or onion by-products. In this study, a two-stage fermentation of OV using Saccharomyces cerevisiae ATCC9763 and Acetobacter pasteurianus CICC20001 was carried out at 28 °C, the titratable acidity reached 4.01%, and the Y A/E was 69.64% at 72 h. Based on this, semi-continuous fermentation was performed, proceeded to charge-discharge consisting of three cycles, and the yield, productivity, and specific production rate were 76.71%, 17.73 g/(L·d), and 20.51 h−1, respectively, which was higher than fed-batch fermentation. The in vivo antioxidant experiments showed that OV significantly increased GSH-Px, SOD, and CAT enzyme activities of Caenorhabditis elegans at 271.57, 129.26, and 314.68%, respectively. Nutritional analysis revealed that the total flavonoids and polyphenols were 3.01 mg/mL and 976.76 µg/mL, respectively. It was also shown that the acetic acid to total organic acid (A/T) ratio of OV was 79.02%, and the total free amino acid content was 262.30 mg/100 mL, 1.78–7.44 times higher than other fruit vinegar. The OV prepared in this study showed higher quality than the commercial vinegar.

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.

The Potential of Sequential Fermentations in Converting C1 Substrates to Higher-Value Products.

Today production of (bulk) chemicals and fuels almost exclusively relies on petroleum-based sources, which are connected to greenhouse gas release, fueling climate change. This increases the urgence to develop alternative bio-based technologies and processes. Gaseous and liquid C1 compounds are available at low cost and often occur as waste streams. Acetogenic bacteria can directly use C1 compounds like CO, CO2, formate or methanol anaerobically, converting them into acetate and ethanol for higher-value biotechnological products. However, these microorganisms possess strict energetic limitations, which in turn pose limitations to their potential for biotechnological applications. Moreover, efficient genetic tools for strain improvement are often missing. However, focusing on the metabolic abilities acetogens provide, they can prodigiously ease these technological disadvantages. Producing acetate and ethanol from C1 compounds can fuel via bio-based intermediates conversion into more energy-demanding, higher-value products, by deploying aerobic organisms that are able to grow with acetate/ethanol as carbon and energy source. Promising new approaches have become available combining these two fermentation steps in sequential approaches, either as separate fermentations or as integrated two-stage fermentation processes. This review aims at introducing, comparing, and evaluating the published approaches of sequential C1 fermentations, delivering a list of promising organisms for the individual fermentation steps and giving an overview of the existing broad spectrum of products based on acetate and ethanol. Understanding of these pioneering approaches allows collecting ideas for new products and may open avenues toward making full use of the technological potential of these concepts for establishment of a sustainable biotechnology.

The Influence of Sugar Composition and pH Regulation in Batch and Continuous Acetone–Butanol–Ethanol Fermentation

Acetone–butanol–ethanol (ABE) fermentation is influenced by external conditions. This work aimed to study the influence of pH regulation on monosaccharide composition in batch and continuous fermentation processes to determine butanol production and productivity. Batch fermentations with ammonium acetate or calcium carbonate combined with minimum pH control (pH ≥ 4.8 or 5.1) were assessed with pure xylose and glucose/xylose mixtures (ratios of 1:1 and 3:1). Continuous two-stage fermentation was developed using plastic rings to retain the biomass. Although batch fermentations with pure xylose performed better without active minimum pH control with both buffers, minimum pH control was necessary to metabolize xylose in the presence of glucose. Xylose uptake was favored by the use of calcium carbonate and pH ≥ 5.1 at a ratio of 1:1, while ammonium acetate and a pH ≥ 4.8 was the best option for a 3:1 ratio. The best butanol production and productivity values with sugar mixtures in batch reactors were 8.8 g L−1 and 0.61 g L−1 h−1 with an ammonium acetate pH ≥ 4.8 (ratio 3:1). The glucose/xylose ratio combined with pH regulation thus modulated xylose metabolism and solvent production in batch modes. Immobilized cells combined with operating at D = 0.333 h−1 and pH regulation increased butanol productivity almost fourfold up to 2.4 ± 0.2 g L−1 h−1.

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%.

Effects of fermented soybean meal with Bacillus velezensis, Lactobacillus spp. or their combination on broiler performance, gut antioxidant activity and microflora.

A series of experiment were conducted to evaluate the effects of replacing a part of soybean meal (SBM) at 6% of broiler diets with fermented soybean meal (FSBM) obtained by single or two-stage fermentation by measuring growth performance, antioxidant activity in the jejunum and distal intestinal microflora. Soybean meal samples were prepared by single-stage fermentation using Bacillus velezensis (Bv) (FSBMB), or Lactobacillus spp. (as commercial control) (FSBML). Additional SBM sample was prepared by two-stage fermentation using Bv and subsequently using Lactobacillus brevis ATCC 367 (Lb) (FSBMB+L). Enzyme activity, chemical composition, trichloroethanoic acid-nitrogen solubility index (TCA-NSI) and antioxidant activity were measured. Then, in an in vivo study, 320 Ross308 broilers were divided into four groups with ad libitum supply of feed and water. Four groups were fed either a corn-soybean meal diet (SBM), or one of fermented SBM diets (FSBMB+L, FSBMB, and FSBML). Growth, serum characteristics, microflora, and the mRNA expression of selected genes were measured. Compared to SBM, FSBMB+L contained lower galacto-oligosaccharide, allergic protein, and trypsin inhibitor, and higher TCA-NSI by about three times (p<0.05). Reducing power and 1,1-diphenyl-2-picrylhydrazyl free radical scavenging ability correlated positively with the TCA-NSI content in FSBM. Growth performances were not significantly different among four groups. In jejunum of 35-day-old broilers, partial replacement of SBM by FSBMB+L increased the activity of superoxide dismutase and catalase (CAT), and the FSBMB group had the highest catalase activity (p<0.05). Partial replacement of SBM by FSBM increased relative mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and peptide transporter 1 (PepT1) (p<0.05); however, FSBMB+L increased CAT mRNA level to 5 times of the control (p<0.05). Using Bv- and Lb-processed SBM through two-stage fermentation to partially replace 6% of diets will improve the gut's antioxidant activity under commercial breeding in broilers.

Bio-isopropanol production in Corynebacterium glutamicum: Metabolic redesign of synthetic bypasses and two-stage fermentation with gas stripping

Isopropanol is a commodity chemical widely used as a biofuel, fuel additive, rubbing alcohol and intermediate in various fields. Here, an engineered Corynebacterium glutamicum overproducing isopropanol was developed. To our knowledge, despite a representative industrial host to produce valuable chemicals, the high-level production of isopropanol in C. glutamicum has never been reported. First, the problem of the inability to produce isopropanol was solved by finding a key factor in its metabolism. The consolidation and modular optimization of synthetic bypasses including succinate and mevalonate bypasses enhanced isopropanol production. Flux redistribution of central metabolism significantly directed the carbon flux toward isopropanol biosynthesis. The final engineered strain produced 10.25 ± 1.12 g/L isopropanol in two-stage fed-batch fermentation with an optimized gas stripping, which is the highest titer, yield and productivity in C. glutamicum. These strategies could be useful for the high-level production of isopropanol in C. glutamicum.

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.

Onion Vinegar Quality Evaluation and its Alleviate Oxidative Stress Mechanism in Caenorhabditis elegans Via SKN-1

Recently, there has been renewed interest in biorefining of agricultural onion into functional products. In this study, onion vinegar (OV) are prepared by a two-stage semi-continuous fermentation method, and its content of total flavonoids (3.01 mg/mL) and polyphenols (976.76 μg/mL) is superior to other commercial vinegars. OV possesses a high radical scavenging activity and enhances the antioxidant enzyme activities in vivo, alleviating intracellular oxidative stress in Caenorhabditis elegans. Treated by OV, the 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH·), diammonium 2,2′-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS+·) and 2-phenyl-4,4,5,5- tetramethylimidazoline-1-oxyl 3-Oxide (PTIO·) free radicals clearance rates are 88.76, 98.76 and 90.54%, respectively in vitro. Whereas the glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) enzyme activities in C. elegans reach 271.57, 129.26, and 314.68%, respectively. Using RNAi and RT-PCR, it has been further confirmed that OV modulates transcription factor SKN-1, the nuclear factor erythroid 2-related factor 2 (Nrf2) homologous, in C. elegans, enhancing the resistance of C. elegans against sodium arsenite stress. Lifespan analysis reveals that 1 mL OV extends the maximum lifespan of the nematode to 26 days. Evidence is presented which shows that OV increases the lifespan of C. elegans by activating the SKN-1 signaling pathway. Overall, the OV is a well functional condiment, enhancing the value-added of onion.

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.

Effects of Microbial Transformation on the Biological Activities of Prenylated Chalcones from Angelica keiskei.

Microbial transformation is an alternative method for structural modification. The current study aimed at application of microbial transformation for discovering new derivatives and investigating the structure-activity relationship of isobavachalcone (1), 4-hydroxyderricin (2), and xanthoangelol (3) isolated from the herb Angelica keiskei. In the initial screening process, 1–3 were incubated with microbes using a two-stage fermentation method and analyzed through TLC monitoring. The screening results showed that Rhizopus oryzae and Mucor hiemalis were able to transform 1 and 2, respectively. Additionally, M. hiemalis and Mortierella ramanniana var. angulispora were able to transform 3. Following scale-up fermentation, four new (4, 5, 7, and 10) and five known (6, 8, 9, 11, and 12) metabolites were produced. Cytotoxicity of all the compounds (1–12) was investigated using three human cancer cell lines including A375P, HT-29, and MCF-7 by MTT method. Meanwhile, the tyrosinase inhibitory activity of 1–12 was evaluated using l-tyrosine as a substrate. Overall, 1 and 3 displayed the highest cytotoxicity, and 5 and 7 exhibited the most potent tyrosinase inhibitory activity with relatively low cytotoxicity. This allowed us to postulate that the introduction of 4′-O-glucopyranosyl group led to the reduction in cytotoxicity and improvement in tyrosinase inhibitory activity.

Assessing the potential of Schizochytrium sp. HX-308 for microbial lipids production from corn stover hydrolysate.

Schizochytrium sp. has received increasing attention as promising commercial resource for the sustainable production of lipids, due to their fast growth rate and high lipid content. However, the price of glucose represents a significant proportion of the total substrate cost. Therefore, in this study, the lignocellulosic hydrolysate of corn stover hydrolysate (CSH) was used as low-cost culture medium to replace glucose in Schizochytrium sp. fermentation. When Schizochytrium sp. HX-308 was fermented with 20% glucose from CSH and 80% of glucose from pure glucose, the lipid production reached 21.2gL-1 , which is lower than that of using 100% of pure glucose. However, the shifts of fatty acid composition indicated that CSH has great potential to enhance the percentage of polyunsaturated fatty acids (PUFAs) in total lipids. However, as the second largest carbon source in CSH, xylose was not utilized by the Schizochytrium sp. HX-308, and further analysis showed that probably because it does not possess a functional xylulose kinase. In addition, the degradation products in lignocellulosic hydrolysate have a strong inhibitory effect on cell growth, so it is necessary to investigate the tolerance of Schizochytrium sp. HX-308 to degradation products. Here, the effects of five typical degradation products on the growth and lipid synthesis were further investigated. Schizochytrium sp. HX-308 showed good tolerance to furan derivatives and organic acids, but low tolerance to phenolic compounds. Furthermore, in order to improve the lipid accumulation using CSH, the two-stage fermentation strategy was developed, resulting in a 54.8% increase compared to that of the one-stage strategy. In summary, this study provides a reference for further fermentation engineering with cheap lignocellulosic biomass as substrate.

Development of in silico strategies to photoautotrophically produce poly-β-hydroxybutyrate (PHB) by cyanobacteria

Poly(3-hydroxybutyrate) (PHB), a biopolymer similar to polypropylene, is currently produced by heterotrophic bacteria. However, the elevated cost of raw material used as carbon and energy sources are promoting the development of new sustainable bioprocess based on photoautotrophic routes costs. In unbalanced growth conditions, some cyanobacteria can accumulate PHB using CO2 as the sole carbon source, becoming an option for PHB production. In this work, we study in silico PHB production with the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). We consider two different strategies for in silico strain design: coupled growth-PHB production and a two-stage fermentation strategy. For both strategies we consider a GEnome-scale Model (GEM) of Synechocystis, which was modified to account for PHB production. The coupled approach relies on a bilevel optimization problem that identifies gene knock-outs needed to obtain a mutant that couples PHB production with biomass formation. For this case, two mutants that successfully achieve the required coupling are obtained, and the best one is analyzed in terms of intervention strategies, flux distributions and productivities. For the two-stage fermentation strategy, we consider two bioreactors in series. The first one is optimized for biomass production under balanced growth conditions, and the second one is optimized for PHB production under nitrogen starvation conditions. Both stages are simulated within dfba, a python tool for solving the Dynamic Flux Balance Analysis (DFBA) problem which provides the solution of a bioreactor model subject to the mass balances of the GEM. Numerical results for the two-stage approach provide a PHB concentration of 4.764 g PHB/L while the mutant provides a lower concentration of 0.391 g PHB/L. Nevertheless, both strategies provide high PHB content per cell dry weight (cdw), which makes results attractive for photosynthetic PHB production.

Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis

Squalene is an important polyunsaturated triterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Currently, the main method for squalene production is extraction from oil-producing plants, but the scale is limited. The microbial fermentation with Saccharomyces cerevisiae still needs improvement to be economically viable. This study aimed to improve squalene production by metabolic engineering and random mutagenesis. First, the mevalonate (MVA) pathway was enhanced, by integrating tHMG1 and IDI1 into multi-copy site Ty2. Subsequently, the ACL gene from Yarrowia lipolytica, encoding citrate lyase was introduced and the β-oxidation pathway was enhanced with multiple copies of key genes. In addition, a high throughput screening strategy based on Nile red staining was established for high squalene-producer screening. After treatment with ARTP mutagenesis, a higher-producing mutant was obtained, with squalene production enhanced by 18.4%. A two-stage fermentation of this mutant in a 5 L bioreactor produced 8.2 g/L of squalene. These findings may facilitate the development of industrial squalene production by fermentation and potentially, other terpenoids.

Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly, protein extraction methods are predominantly restricted to chemical methods, and mechanical methods reliant on expensive specialized equipment more tuned for large-scale applications. In this article, we outline detailed protocols for the use of an engineered autolysis/autohydrolysis E. coli strain, in two-stage fermentations in shake-flasks. This two-stage fermentation protocol does not require optimization of expression conditions and results in high protein titers. Cell lysis in an engineered strain is tightly controlled and only triggered post-culture by addition of a 0.1% detergent solution. Upon cell lysis, a nuclease digests contaminating host oligonucleotides, which facilitates sample handling. This method has been validated for use at different scales, from microtiter plates to instrumented bioreactors. Graphic abstract: Two-stage protein expression, cell autolysis and DNA/RNA autohydrolysis. Reprinted with permission from Menacho-Melgar et al. (2020a). Copyright 2020 John Wiley and Sons.

Two-Stage Fermentation Enhanced Single-Cell Protein Production by Yarrowia Lipolytica from Food Waste

Two-Stage Fermentation Enhanced Single-Cell Protein Production by Yarrowia Lipolytica from Food Waste

Two-Stage Fermentation of Corn and Soybean Meal Mixture by Bacillus Subtilis and Lactobacillus Acidophilus: Optimization, Physicochemical Property and Microbial Community

Two-Stage Fermentation of Corn and Soybean Meal Mixture by Bacillus Subtilis and Lactobacillus Acidophilus: Optimization, Physicochemical Property and Microbial Community

Hydrogen fermentation by photosynthetic bacteria mixed culture with silicone immobilization and metagenomic analysis

The biohydrogen production is promising for the alternative green hydrogen. The two-stage of the dark and photo-fermentation system increases the hydrogen production yield. In this study, the experiments carried out the cell immobilizations on dark and photo bacteria; and liquid suspended photo bacteria for those two-stage batch systems. The optimized parameters obtained, such as materials for immobilized cells, different substrates, and the substrate concentrations for hydrogen fermentation.The experiment results showed pure substrate glucose could produce a higher amount of hydrogen in the photo immobilized cells, the hydrogen concentration was up to about 40%, the utilization rate of glucose was as high as 99.41%, the accumulated hydrogen volume was 169.13 mL, and the hydrogen yield, HY, was 0.63 mol H2/mol glucose. In addition, the activated immobilized cells are used for two-stage fermentation to generate hydrogen, HY 1.75 mol H2/mol substrate. The two-stage batch fermentations for producing hydrogen, used the Gompertz equation model to obtain the cumulative hydrogen volume and hydrogen production rate (HPR). When the ratio of dark immobilized cells (DIMC) to photo suspended liquids (PSL) is 1.0:5.0, the hydrogen production shows well performance.Moreover, an advanced molecular biological technique, next-generation sequencing (NGS) with ultra-high-throughput DNA output, was applied to perform microbial communities in a different stage of hydrogen fermentation. The NGS help to understand the detailed microbial community change in such fermentation processes and provide better biological efficacy via clarifying the role of microorganisms and their interactions. After the experiments, the Clostridia increased to 28%, and the Enterobacter also increased to 15%, therefore the Rhodobacter and Bosea were reduced from 55% to 17%. Understand the detailed composition of microbial populations in stages of such bio-system will provide the useful information via clarify the biological role of microorganisms and its interactions.

Sequential Production of ᴅ-xylonate and Ethanol from Non-Detoxified Corncob at Low-pH by Pichia kudriavzevii via a Two-Stage Fermentation Strategy.

Improving the comprehensive utilization of sugars in lignocellulosic biomass is a major challenge for enhancing the economic viability of lignocellulose biorefinement. A robust yeast Pichia kudriavzevii N-X showed excellent performance in ethanol production under high temperature and low pH conditions and was engineered for ᴅ-xylonate production without xylitol generation. The recombinant strain P. kudriavzevii N-X/S1 was employed for sequential production of ᴅ-xylonate and ethanol from ᴅ-xylose, feeding on ᴅ-glucose without pH control in a two-stage strategy of aerobic and shifting micro-aerobic fermentation. Acid-pretreated corncob without detoxification and filtration was used for ᴅ-xylonate production, then simultaneous saccharification and ethanol fermentation was performed with cellulase added at pH 4.0 and at 40 °C. By this strategy, 33.5 g/L ᴅ-xylonate and 20.8 g/L ethanol were produced at yields of 1.10 g/g ᴅ-xylose and 84.3% of theoretical value, respectively. We propose a promising approach for the sequential production of ᴅ-xylonate and ethanol from non-detoxified corncob using a single microorganism.