Efficient Fermentation Process Research Articles

Integrated strain- and process design enable production of 220\xa0g\xa0L\u22121 itaconic acid with Ustilago maydis

BackgroundItaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L−1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production.ResultsIn this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L−1, with a total acid titer of 248 g L−1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus.ConclusionIn this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

A review on characteristics of food waste and their use in butanol production.

Biobutanol offers several advantages and a larger market, that make it a biofuel to be studied with great interest. In fact, butanol has an energy content similar to that of gasoline, and it can be used as an alternative fuel to gasoline. It is a biofuel that is safe for the environment. The optimization of the production of butanol thus appears as an attractive option. Butanol production from food waste (FW) is a process for carbon recovery and a method for solid waste recycling. Recently, the use of FW and food processing waste (FPW) as raw material for the production of butanol has attracted much interest. However, an efficient fermentation process is vital to improve the production of biobutanol. To the best of our knowledge, no review on butanol production from FW has been presented so far. Thus, this review focuses on the characteristics of FW and its potential to produce butanol. In addition, the main factors that affect their use for the production of butanol are also discussed.

Evolution toward the utilization of mango leaves as lignocellulosic material in bioethanol production: A review of process parameter and integrated technologies

AbstractRecently, the production of bioethanol is shifted to secondary bioethanol which is produced from nonedible lignocellulosic feedstock to avoid the food versus fuel issue. Mango leaves, a kind of nonedible lignocellulosic material (LCM) that possess a relatively 80.7% of holocellulose (inclusive of cellulose and hemicellulose), appear to be potential candidate to serve as cheap substrate source for bioethanol production. Hence, the objective of this article is to present the current scenario and the potential of mango leaves as a substrate source for bioethanol production. This article also provides an overview on various process parameters such as temperature, pH, substrate concentration, and incubation time that required to be optimized for an efficient fermentation process in the bioethanol production from LCM. Apart from that, several integrated fermentation technologies in bioethanol production which include separate hydrolysis and fermentation, simultaneous saccharification and fermentation, simultaneous saccharification and co‐fermentation, and consolidated bioprocessing will also be discussed in this article. Based on the findings, it is clear that mango leaves have the potential to serve as feedstock for bioethanol production.

Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: What to expect from anaerobic fungus Orpinomyces sp.

Energy-efficient biogas reactors are often designed and operated mimicking natural microbial ecosystems such as the digestive tracts of ruminants. Anaerobic fungi play a crucial role in the degradation of lignocellulose-rich fiber thanks to their high cellulolytic activity. Fungal bioaugmentation is therefore at the heart of our understanding of enhancing anaerobic digestion (AD). The efficiency of bioaugmentation with anaerobic fungus Orpinomyces sp. was evaluated in lignocellulose-based AD configurations. Fungal bioaugmentation increased the methane yield by 15–33% during anaerobic co-digestion of cow manure and selected cereal crops/straws. Harvesting stage of the crops was a decisive parameter to influence methane production together with fungal bioaugmentation. A more efficient fermentation process in the bioaugmented digesters was distinguished by relatively-higher abundance of Synergistetes, which was mainly represented by the genus Anaerobaculum. On the contrary, the composition of the methanogenic archaea did not change, and the majority of methanogens was assigned to Methanosarcina.

Advances and opportunities in biomass conversion technologies and biorefineries for the development of a bio-based economy

The desire to have a more sustainable future, with lower emissions of carbon and sulfur to the atmosphere, a more appropriate reuse and valorization of wastes, and less dependency on oil has motivated the society to develop processes where renewable biomass is used as a feedstock for the production of fuels, chemicals, energy and materials. In addition, a bio-based economy has also potential to generate new jobs and new opportunities for entrepreneurship, with further benefits to the society. In view of this, great efforts have been done in order to develop efficient, sustainable and cost competitive bio-based processes able to be implemented in industrial scale. Although important advances were achieved and some processes are already available in a large scale, improvements are still needed to have a final product at a more competitive market price. In this sense, the strategy of integrating biorefineries to produce a variety of products from biomass has been considered as an important alternative to improve the financial performance. This paper highlights the most recent advances and opportunities in biomass conversion technologies and biorefineries for the development of a bio-based economy. Technological aspects including the hemicellulose integration and use of sugars for different products, lignin valorization, development of efficient and low-cost pretreatment technologies and development of highly efficient fermentation processes are also presented and discussed.

Substrate sustained release-based high efficacy biosynthesis of GABA by Lactobacillus brevis NCL912

BackgroundGamma-aminobutyric acid (GABA) plays a significant role in the food and drug industries. Our previous study established an efficient fed-batch fermentation process for Lactobacillus brevis NCL912 production of GABA from monosodium l-glutamate; however, monosodium l-glutamate may not be an ideal substrate, as it can result in the rapid increase of pH due to decarboxylation. Thus, in this study, l-glutamic acid was proposed as a substrate. To evaluate its potential, key components of the fermentation medium affecting GABA synthesis were re-screened and re-optimized to enhance GABA production from L. brevis NCL912.ResultsThe initial fermentation medium (pH 3.3) used for optimization was: 50 g/L glucose, 25 g/L yeast extract, 10 mg/L manganese sulfate (MnSO4·H2O), 2 g/L Tween-80, and 220 g/L l-glutamic acid. Glucose, a nitrogen source, magnesium, and Tween-80 had notable effects on GABA production from the l-glutamic acid-based process; other factors showed no or marginal effects. The optimized levels of the four key components in the fermentation medium were 25 g/L glucose, 25 g/L yeast extract FM408, 25 mg/L MnSO4·H2O, and 2 g/L Tween-80. A simple and efficient fermentation process for the bioconversion of GABA by L. brevis NCL912 was subsequently developed in a 10 L fermenter as follows: fermentation medium, 5 L; glutamic acid, 295 g/L; inoculum, 10% (v/v); incubation temperature, 32 °C; and agitation, 100 rpm. After 48 h of fermentation, the final GABA concentration increased up to 205.8 ± 8.0 g/L.Conclusionsl-Glutamic acid was superior to monosodium l-glutamate as a substrate in the bioproduction of GABA. Thus, a high efficacy bioprocess with 205 g/L GABA for L. brevis NCL912 was established. This strategy may provide an alternative for increasing the bioconversion of GABA.

Extracellular Phytase Production by the Wine Yeast S. cerevisiae (Finarome Strain) during Submerged Fermentation

One of the key steps in the production of phytases of microbial origin is selection of culture parameters, followed by isolation of the enzyme and evaluation of its catalytic activity. It was found that conditions for S. cerevisiae yeast culture, strain Finarome, giving the reduction in phytic acid concentration of more than 98% within 24 h of incubation were as follows: pH 5.5, 32 °C, continuous stirring at 80 rpm, the use of mannose as a carbon source and aspartic acid as a source of nitrogen. The highest catalytic activity of the isolated phytase was observed at 37 °C, pH 4.0 and using phytate as substrate at concentration of 5.0 mM. The presence of ethanol in the medium at a concentration of 12% v/v reduces the catalytic activity to above 60%. Properties of phytase derived from S. cerevisiae yeast culture, strain Finarome, indicate the possibility of its application in the form of a cell’s free crude protein isolate for the hydrolysis of phytic acid to improve the efficiency of alcoholic fermentation processes. Our results also suggest a possibility to use the strain under study to obtain a fusant derived with specialized distillery strains, capable of carrying out a highly efficient fermentation process combined with the utilization of phytates.

New Approach of Highly Efficient Fermentation Process for Bioethanol using Xylose as Agriculture Residues

Agricultural waste biomass has already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR) and the protein engineered xylitol dehydrogenase (XDH) (with NADP) of Pichia stipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomyces cerevisiae as judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARS strain, in which NADP+-dependent XDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH.

New Approach of Highly Efficient Fermentation Process for Bio ethanol using Xylose as Agricultur e Residues

Agricultural waste biomasshas already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR)and the protein engineered xylitol dehydrogenase (XDH)(with NADP) of Pichiastipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomycescerevisiaeas judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARSstrain, in which NADP+-dependentXDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH.

Development of an Efficient Fermentation Process by Response Surface Methodology for Enhanced Dextransucrase Production from Leuconostoc lactis KU665298

Development of an Efficient Fermentation Process by Response Surface Methodology for Enhanced Dextransucrase Production from Leuconostoc lactis KU665298

Improvement of (R,R)-2,3-butanediol production from corn stover hydrolysate by cell recycling continuous fermentation

To achieve industrial (R,R)-2,3-butanediol ((R,R)-2,3-BDL) production from various lignocellulosic biomass, it is essential to develop an efficient fermentation process to overcome the challenge of carbon catabolite repression (CCR). The present study comprehensively investigated the effects of different glucose/xylose mixtures and fermentation strategies on (R,R)-2,3-BDL production using a nonpathogenic strain of Paenibacillus polymyxa. In both batch and fed-batch mode, we observed slow sugar utilization and a lower productivity. By employing cell-recycling continuous fermentation system for (R,R)-2,3-BDL production from mixed sugar, the volumetric productivity was significantly increased, by an average of 2-fold higher compared to alternative fermentation modes. Moreover, the (R,R)-2,3-BDL titer, volumetric productivity and yield achieved 18.80g/l, 1.13g/l/h and 0.313g/g when corn stover hydrolysates (CSH)was used as substrate. The results suggested that the reported fermentation system could efficiently eliminate CCR to improve (R,R)-2,3-BDL productivity. Thus, it had great potential for the industrial-scale (R,R)-2,3-BDL production from lignocellulosic hydrolysates.

Non-sterile fermentations for the economical biochemical conversion of renewable feedstocks.

Heavy reliance on petroleum-based products drives continuous exploitation of fossil fuels, and results in serious environmental and climate problems. To address such an issue, there is a shift from petroleum sources to renewable ones. Biochemical conversion via fermentation is a primary platform for converting renewable sources to biofuels and bulk chemicals. In order to provide cost-competitive alternatives, it is imperative to develop efficient, cost-saving, and robust fermentation processes. Non-sterile fermentation offers several benefits compared to sterile fermentation, including elimination of sterility, reduced maintenance requirements, relatively simple bioreactor design, and simplified operation. Thus, cost effectiveness of non-sterile fermentation makes it a practical platform for low cost, large volume production of biofuels and bulk chemicals. Many approaches have been developed to conduct non-sterile fermentation without sacrificing the yields and productivities of fermentation products. This review focuses on the strategies for conducting non-sterile fermentation. The challenges facing non-sterile fermentation are also discussed.

Butanol fermentation of the brown seaweed Laminaria digitata by Clostridium beijerinckii DSM-6422

Seaweed represents an abundant, renewable, and fast-growing biomass resource for 3rd generation biofuel production. This study reports an efficient butanol fermentation process carried out by Clostridium beijerinckii DSM-6422 using enzymatic hydrolysate of the sugar-rich brown seaweed Laminaria digitata harvested from the coast of the Danish North Sea as substrate. The highest butanol yield (0.42g/g-consumed-substrates) compared to literature was achieved, with a significantly higher butanol:acetone-butanol-ethanol (ABE) molar ratio (0.85) than typical (0.6). This demonstrates the possibility of using the seaweed L. digitata as a potential biomass for butanol production. For the first time, consumption of alginate components was observed by C. beijerinckii DSM-6422. The efficient utilization of sugars and lactic acid further highlighted the potential of using this strain for future development of large-scale cost-effective butanol production based on (ensiled) seaweed.

Optimization of critical medium components for enhancing antibacterial thiopeptide nocathiacin I production with significantly improved quality

Nocathiacin I, a glycosylated thiopeptide antibiotic, displays excellent antibacterial activities against multidrug resistant bacterial pathogens. Previously, a novel nocathiacin I formulation for intravenous administration has been successfully developed and its aqueous solubility is greatly enhanced for clinical application. The purpose of the present study was to increase the fermentation titer of nocathiacin I and reduce or eliminate analogous impurities by screening the medium ingredients using response surface methodology. After a sysmatic optimization, a water-soluble medium containing quality-controllable components was developed and validated, resulting in an increase in the production of nocathiacin I from 150 to 405.8 mg·L−1 at 150-L scale. Meanwhile, the analogous impurities existed in reported processes were greatly reduced or eliminated. Using optimized medium for fermentation, nocathiacin I with pharmaceutically acceptable quality was easily obtained with a recovery of 67%. In conclusion, the results from the present study offer a practical and efficient fermentation process for the production of nocathiacin I as a therapeutic agent.

P209 水素・メタン二段醗酵による有機性廃棄物処理

P209 水素・メタン二段醗酵による有機性廃棄物処理

3-10.有機性廃棄物の高効率水素・メタン醗酵を中心とした二段醗酵技術研究開発((3)微生物等1,Session 3 バイオマス等)

3-10.有機性廃棄物の高効率水素・メタン醗酵を中心とした二段醗酵技術研究開発((3)微生物等1,Session 3 バイオマス等)

Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae.

During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the α-keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources.IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines.

Betaine Improves Polymer-Grade D-Lactic Acid Production by Sporolactobacillus inulinus Using Ammonia as Green Neutralizer.

The traditional CaCO3-based fermentation process generates huge amount of insoluble CaSO4 waste. To solve this problem, we have developed an efficient and green D-lactic acid fermentation process by using ammonia as neutralizer. The 106.7 g/L of D-lactic acid production and 0.89 g per g of consumed sugar were obtained by Sporolactobacillus inulinus CASD with a high optical purity of 99.7% by adding 100 mg/L betaine in the simple batch fermentation process. The addition of betaine was experimentally proven to protect cell at high concentration of ammonium ion, increase the D-lactate dehydrogenase specific activity and thus promote the production of D-lactic acid.

Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol.

We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan5 strain to prevent hyperglycosylation. The two (His)6-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15mgL-1 of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man5GlcNAc2 with, however, a significant contribution of Man4-10GlcNAc2. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O2) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.

Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

ABSTRACTUsing a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3 L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17 g/L), corn steep liquor (69.25 g/L), and casamino acids (80.68 g/L) in a stirred tank bioreactor under operating conditions constant at 635 rpm, a temperature of 26°C, and pH 3.3 produced 1.25 × 108 blastospores (bls)/ml, with 93% viability after 120 h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.