Development Of Fermentation Process Research Articles

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 μg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

Reassessing the out-of-phase phenomenon in shake flasks by evaluating the angle-dependent liquid distribution relative to the direction of the centrifugal acceleration.

Shake flasks are still the most relevant experimental tool in the development of viscous fermentation processes. The phase number, which defines the onset of the unfavorable out-of-phase (OP) phenomenon in shake flasks, was previously defined via specific power input measurements. In the OP state, the bulk liquid no longer follows the orbital movement of the imposed centrifugal force, which is for example, detrimental to oxygen transfer. In this study, an optical fluorescence technique was used to measure the three-dimensional liquid distribution in shake flasks. Four new optically derived evaluation criteria for the phase transition between the in-phase and OP condition were established: (a) thickness of the liquid film left on the glass wall by the rotating bulk liquid, (b) relative slope of the leading edge of bulk liquid (LB) lines, (c) trend of the angular position of LB, and (d) very high angular position of the leading edge. In contrast to the previously applied power input measurements, the new optical evaluation criteria describe the phase transition in greater detailed. Instead of Ph = 1.26, a less conservative value of Ph = 0.91 is now suggested for the phase transfer, which implies a broader operating window for shake flask cultivations with higher viscosities.

Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii.

Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in A. kawachiiIMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

Primary and Secondary Metabolic Effects of a Key Gene Deletion (ΔYPL062W) in Metabolically Engineered Terpenoid-Producing Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion of YPL062W in S. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function of YPL062W and the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered that YPL062W deletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed that YPL062W functions as a core promoter for ALD6 and that the expression level of ALD6 is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062W increases the production of all major terpenoid classes (C10, C15, C20, C30, and C40). Our study not only elucidated the biological function of YPL062W but also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCE Although computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms of YPL062W deletion in S. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function of YPL062W, demonstrates a strong correlation between YPL062W and terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.

Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression of β-mannanase has been reported in Pichia pastoris under control of the GAP promoter. Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature. The aim of this research was to improve the expression level of β-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature. To this end, a panel of strains harboring different copy numbers of β-mannanase gene were obtained by multiple zeocin concentration gradients screening, the influence of gene copy number on the expression of β-mannanase in Pichia pastoris X33 was investigated. With the constitutive GAP promoter, the four copies strain exhibited a 4.04-fold higher β-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain, but an increase of the copy number above four resulted in a decrease of expression. Furthermore, the effects of culture temperature were studied in flask. The decreased culture temperature of four copies strain resulted in a 1.8-fold (26 °C) and 3.5-fold (22 °C) higher β-mannanase activity compared to that at 30 °C. A fed-batch strategy was successfully used for high cell-density fermentation and β-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.

A small‐scale investigation process for the Clostridium acetobutylicum production of butanol using high‐energy carbon heavy ion irradiation

Applied heavy ion irradiation technology and butanol industrial practices as a whole have been used as a strategy for the development of an attractive alternative to petroleum-based fuels. Clostridium acetobutylicum (C. acetobutylicum) strains are well documented as fermentation strains for the production of biobutanol. However, it has been reported that solvent production inhibits the growth of these strains, and the accumulation of acetate also inhibits biomass synthesis, rendering the production of butanol from acetone-butanol-ethanol (ABE) fermentation processes economically challenging. In this manuscript, we propose the use of high-energy carbon heavy ion irradiation from the Heavy Ion Research Facility in Lanzhou (HIRFL) to obtain a culture with an increased butanol yield. Our findings suggest that the use of a high-energy 12C6+ heavy ion irradiation dose of 45 Gy with an energy of 135AMeV and ion pulses/levels of 106-108 favours ABE solvent production in an irradiated strain compared with the non-irradiated strain. The strategy reported in this manuscript may contribute to the development of a cost-effective butanol fermentation process that is competitive with similar fermentation processes.

Liquid-liquid equilibria for (volatile fatty acids + water + alcohol ethoxylates): Experimental measurement of pseudo-ternary systems

Abstract Experiments were conducted to establish the phase diagrams for ternary systems containing (acetic acid or propionic acid or butyric acid + water + alcohol ethoxylate surfactant) at 308.15 K and ambient pressure. Results indicated that the systems belong to Type 1 Treybal classification with water–surfactant binary pair exhibiting partial miscibility. The liquid–liquid equilibrium data were used to determine the ability of the surfactant to extract the acids from aqueous solutions. The calculated distribution coefficients and separation factors indicated that the effectiveness of the surfactant to extract acids follow the trend: butyric acid > propionic acid > acetic acid. The effectiveness of the surfactant to extract butyric acid from aqueous solutions is similar to previously studied environmentally-friendly solvents with added benefit of being non-inhibitory to microbial growth and metabolism. These characteristics of the alcohol ethoxylate used in this study could be exploited for the development of an extractive butyric acid fermentation process.

Free lactic acid production under acidic conditions by lactic acid bacteria strains: challenges and future prospects.

Lactic acid (LA) is an important platform chemical due to its significant applications in various fields and its use as a monomer for the production of biodegradable poly(lactic acid) (PLA). Free LA production is required to get rid of CaSO4, a waste material produced during fermentation at neutral pH which will lead to easy purification of LA required for the production of biodegradable PLA. Additionally, there is no need to use corrosive acids to release free LA from the calcium lactate produced during neutral fermentation. To date, several attempts have been made to improve the acid tolerance of lactic acid bacteria (LAB) by using both genome-shuffling approaches and rational design based on known mechanisms of LA tolerance and gene deletion in yeast strains. However, the lack of knowledge and the complexity of acid-tolerance mechanisms have made it challenging to generate LA-tolerant strains by simply modifying few target genes. Currently, adaptive evolution has proven an efficient strategy to improve the LA tolerance of individual/engineered strains. The main objectives of this article are to summarize the conventional biotechnological LA fermentation processes to date, assess their overall economic and environmental cost, and to introduce modern LA fermentation strategies for free LA production. In this review, we provide a broad overview of free LA fermentation processes using robust LAB that can ferment in acidic environments, the obstacles to these processes and their possible solutions, and the impact on future development of free LA fermentation processes commercially.

Development of an innovative two-stage fermentation process for high-calorific biogas at elevated pressure

The two-stage high-pressure fermentation (HPF) process enables the production of methane at high operating pressure. Pressure significantly reduces the energy needed for injecting the produced biogas into the gas grid by 45–60%. It also allows for incorporating large parts of the necessary biogas upgrading process into the synthesis step. As a result, the two-stage HPF process provides pressurized biogas with methane volume fraction ranging from 0.75 to 0.94. The pressure is not generated by energy intensive gas compression, but in-situ by microbial gas production. In comparison to conventional biomethane production, the overall costs could be reduced up to 20%. HPF is most beneficial when its operating pressure is adapted to that of the gas grid.The article presents briefly the development of the two-stage HPF beginning with tests in batch reactors, followed by experiments on gas solubility, and proof-of-concept in continuously operated methanogenesis reactors (MR) up to 9 bar. It also represents the effect of incorporating microfiltration (MF) of the feed stream, on improving the biogas quality and process stability of a continuously operated lab scale HPF process. By linking the MF with the HPF, methane volume fraction in the MR increases from 0.86 to 0.94 at 25 bar. Finally, the simulation and experimental results show good agreement with each other thereby making them a good basis for further optimization of the HPF process.

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

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018.

Succinic Acid: Technology Development and Commercialization

Succinic acid is a precursor of many important, large-volume industrial chemicals and consumer products. It was once common knowledge that many ruminant microorganisms accumulated succinic acid under anaerobic conditions. However, it was not until the discovery of Anaerobiospirillum succiniciproducens at the Michigan Biotechnology Institute (MBI), which was capable of producing succinic acid up to about 50 g/L under optimum conditions, that the commercial feasibility of producing the compound by biological processes was realized. Other microbial strains capable of producing succinic acid to high final concentrations subsequently were isolated and engineered, followed by development of fermentation processes for their uses. Processes for recovery and purification of succinic acid from fermentation broths were simultaneously established along with new applications of succinic acid, e.g., production of biodegradable deicing compounds and solvents. Several technologies for the fermentation-based production of succinic acid and the subsequent conversion to useful products are currently commercialized. This review gives a summary of the development of microbial strains, their fermentation, and the importance of the down-stream recovery and purification efforts to suit various applications in the context of their current commercialization status for biologically derived succinic acid.

Bioproduction of riboflavin: a bright yellow history.

Riboflavin (vitamin B2) is an essential nutrient for humans and animals that must be obtained from the diet. To ensure an optimal supply, riboflavin is used on a large scale as additive in the food and feed industries. Here, we describe a historical overview of the industrial process of riboflavin production starting from its discovery and the need to produce the vitamin in bulk at prices that would allow for their use in human and animal nutrition. Riboflavin was produced industrially by chemical synthesis for many decades. At present, the development of economical and eco-efficient fermentation processes, which are mainly based on Bacillus subtilis and Ashbya gossypii strains, has replaced the synthetic process at industrial scale. A detailed account is given of the development of the riboflavin overproducer strains as well as future prospects for its improvement.

Advanced digital image analysis method dedicated to the characterization of the morphology of filamentous fungus.

Filamentous fungi have a complex morphology that induces fermentation process development issues, as a consequence of viscosity increase and diffusion limitations. In order to better understand the relationship between viscosity changes and fungus morphology during fermentations of Trichoderma reesei, an accurate image analysis method has been developed to provide quantitative and representative data for morphological analysis. This method consisted of a new algorithm called FACE that allowed sharp images to be created at all positions, segmentation of fungus, and morphological analysis using skeleton and topological approaches. It was applied and validated by characterizing samples of an industrial strain of Trichoderma reesei that had or had not been exposed to an extreme shear stress. This method allowed many morphological characteristics to be identified, among which nine relevant criteria were extracted, regarding the impact of shear stress on the fungus and on the viscosity of the fermentation medium.

P-50 海洋微生物を活用した大型藻類の耐塩性メタン発酵プロセスの開発

P-50 海洋微生物を活用した大型藻類の耐塩性メタン発酵プロセスの開発

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in Escherichia coli.

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.

Open Fermentative Production of L-Lactic Acid from Distillers’ Grains by Lactobacillus casei CICC 6056

Distillers’ grains (DG) are potential fermentable sugar substrates for the production of value-added products. This study focused on the development of an open fermentation process for the production of L-lactic acid from DG using Lactobacillus casei CICC 6056. The open fermentation process was feasible, resulting in an L-lactic acid yield similar to that obtained with sterilized fermentation. However, a decrease in pH below 5.0 after 24 h resulted in a poor L-lactic acid concentration of 9.18 g/L and an inferior reducing sugar conversion rate of 53.6%. Therefore, an optimal pH adjustment method was sought. A pH adjustment to 6.5 every 24 h effectively improved the L-lactic acid concentration to 21.3 g/L with a 97.8% reducing sugar conversion rate. Furthermore, the application of a simultaneous saccharification and fermentation process at 50 °C in open condition increased the L-lactic acid concentration to 25.0 g/L. This yield was superior to L-lactic acid fermentation from DG using a separate hydrolysis and fermentation method (21.3 g/L) and from a commercial sugar in batch culture (23.6 g/L). These results demonstrated that L. casei CICC 6056 has great industrial potential as a superior candidate strain for the production of L-lactic acid from DG due to its broad applicability in a wide temperature range (from 37 to 50 °C).

Biomass, strain engineering, and fermentation processes for butanol production by solventogenic clostridia.

Butanol is considered an attractive biofuel and a commercially important bulk chemical. However, economical production of butanol by solventogenic clostridia, e.g., via fermentative production of acetone-butanol-ethanol (ABE), is hampered by low fermentation performance, mainly as a result of toxicity of butanol to microorganisms and high substrate costs. Recently, sugars from marine macroalgae and syngas were recognized as potent carbon sources in biomass feedstocks that are abundant and do not compete for arable land with edible crops. With the aid of systems metabolic engineering, many researchers have developed clostridial strains with improved performance on fermentation of these substrates. Alternatively, fermentation strategies integrated with butanol recovery processes such as adsorption, gas stripping, liquid-liquid extraction, and pervaporation have been designed to increase the overall titer of butanol and volumetric productivity. Nevertheless, for economically feasible production of butanol, innovative strategies based on recent research should be implemented. This review describes and discusses recent advances in the development of biomass feedstocks, microbial strains, and fermentation processes for butanol production.

Engineering cell factories for producing building block chemicals for bio-polymer synthesis.

Synthetic polymers are widely used in daily life. Due to increasing environmental concerns related to global warming and the depletion of oil reserves, the development of microbial-based fermentation processes for the production of polymer building block chemicals from renewable resources is desirable to replace current petroleum-based methods. To this end, strains that efficiently produce the target chemicals at high yields and productivity are needed. Recent advances in metabolic engineering have enabled the biosynthesis of polymer compounds at high yield and productivities by governing the carbon flux towards the target chemicals. Using these methods, microbial strains have been engineered to produce monomer chemicals for replacing traditional petroleum-derived aliphatic polymers. These developments also raise the possibility of microbial production of aromatic chemicals for synthesizing high-performance polymers with desirable properties, such as ultraviolet absorbance, high thermal resistance, and mechanical strength. In the present review, we summarize recent progress in metabolic engineering approaches to optimize microbial strains for producing building blocks to synthesize aliphatic and high-performance aromatic polymers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0411-0) contains supplementary material, which is available to authorized users.

English

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible intracellular polyesters that are accumulated as energy and carbon reserves by bacterial species, under nutrient limiting conditions. Successful large-scale production of PHAs is dependent on three crucial factors, which include the cost of substrate, downstream processing cost, and process development. In this respect, design and implementation of bioprocess strategies for efficient PHA bioconversions enable high PHA concentrations, yields and productivities. Additionally, development of PHA fermentation processes using inexpensive substrates, such as agro-industrial wastes, facilitates further cost reduction, thus benefitting large-scale PHA production. Thus, the aim of this review is to highlight various bioprocess strategies for high production of PHAs and their novel copolymers in relatively large quantities. This review also discusses the application of kinetic analysis and mathematical modelling as important tools for process optimization and thus improvement of the overall process economics for large-scale production of PHAs.