Kris A. Berglund

Effect of Different Carbon Sources on the Production of Succinic Acid Using Metabolically Engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual‐phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large‐scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L−1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g−1). When using xylose as a carbon source, a yield of 0.50 g g−1 was obtained. In the mixed‐sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g−1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g−1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25–40 g L−1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7–6.7 and 0–2.7 g L−1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L−1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.

ATR FTIR spectroscopy for in situ measurement of supersaturation

The current contribution establishes the technical feasibility of Attenuated Total Reflection (ATR) Fourier transform infrared (FTIR) spectroscopy for the in situ measurement of supersaturation in crystallization processes. The approach was inspired by recent advancements in ATR spectroscopy by way of various light transfer systems for remote sensing and by the increasing availability of ATR configurations well suited for remote, in situ measurements. The feasibility of the technique was investigated using a DIPPER-210® immersion probe manufactured by Axiom Analytical, Inc. Initial experiments conducted using aqueous maleic acid proved that ATR FTIR spectroscopy can be successfully employed to measure supersaturation, solubility and the metastable limit, in situ, with sufficient accuracy and precision.

An investigation of the applicability of attenuated total reflection infrared spectroscopy for measurement of solubility and supersaturation of aqueous citric acid solutions

Abstract Currently applied methods for measurement of solubility and supersaturation based on viscometry, refractometry, interferometry and density require the separation of phases prior to measurement. ATR (attenuated total reflection) infrared spectroscopy provides a unique configuration in which the infrared spectrum of a liquid phase can be obtained in a slurry without phase separation. The applicability of the technique was investigated using a micro Circle® open boat cell equipped with a ZnSe (zinc selenide) ATR rod. Experiments conducted with aqueous citric acid proved that ATR infrared spectroscopy can be successfully employed to determine solubility and supersaturation.

Impact of hemicellulose pre-extraction for bioconversion on birch Kraft pulp properties

The combination of hemicellulose extraction with chemical pulping processes is one approach to generate a sugar feedstock amenable to biochemical transformation to fuels and chemicals. Extractions of hemicellulose from silver birch (Betula pendula) wood chips using either water or Kraft white liquor (NaOH, Na(2)S, and Na(2)CO(3)) were performed under conditions compatible with Kraft pulping, using times ranging between 20 and 90 min, temperatures of 130-160 degrees C, and effective alkali (EA) charges of 0-7%. The chips from select extractions were subjected to subsequent Kraft pulping and the refined pulps were made into handsheets. Several metrics for handsheet strength properties were compared with a reference pulp made without an extraction step. This study demonstrated that white liquor can be utilized to extract xylan from birch wood chips prior to Kraft cooking without decreasing the pulp yield and paper strength properties, while simultaneously impregnating cooking alkali into the wood chips. However, for the alkaline conditions tested extractions above pH 10 resulted in low concentrations of xylan. Water extractions resulted in the highest final concentrations of xylan; yielding a liquor without the presence of toxic or inhibitory inorganics and minimal soluble aromatics that we demonstrate can be successfully enzymatically hydrolyzed to monomeric xylose and fermented to succinic acid. However, water extractions were found to negatively impact some pulp properties including decreases in compression strength, bursting strength, tensile strength, and tensile stiffness while exhibiting minimal impact on elongation and slight improvement in tearing strength index.

The structure of aqueous solutions of some dihydrogen orthophosphates by laser Raman spectroscopy

Powdered crystals and pure, aqueous solutions of ammonium, sodium, and potassium dihydrogen orthophosphates in concentrations ranging from 0.01M to supersaturated were investigated at 30°C using la ...

Inhibition of succinic acid production in metabolically engineered Escherichia Coli by neutralizing agent, organic acids, and osmolarity

The economical viability of biochemical succinic acid production is a result of many processing parameters including final succinic acid concentration, recovery of succinate, and the volumetric productivity. Maintaining volumetric productivities >2.5 g L−1 h−1 is important if production of succinic acid from renewable resources should be competitive. In this work, the effects of organic acids, osmolarity, and neutralizing agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3) on the fermentative succinic acid production by Escherichia coli AFP184 were investigated. The highest concentration of succinic acid, 77 g L−1, was obtained with Na2CO3. In general, irrespective of the base used, succinic acid productivity per viable cell was significantly reduced as the concentration of the produced acid increased. Increased osmolarity resulting from base addition during succinate production only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose consumption rate, but decreased succinic acid yield. When using NH4OH productivity completely ceased at a succinic acid concentration of ∼40 g L−1. Volumetric productivities remained at 2.5 g L−1 h−1 for up to 10 h longer when K‐ or Na‐bases where used instead of NH4OH. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be due to increased organic acid concentrations rather than medium osmolarity.

In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop

Abstract Fiber optic Raman spectroscopy combined with a partial least-squares regression model was demonstrated as a monitor of lysozyme concentration during crystallization in a hanging drop experiment in real time. Raman spectral features of the buffer and protein were employed to build the regression model. The use of fiber optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous solutions, results in a powerful noninvasive probe of the changing environment within the solution. Lysozyme concentrations were monitored in experiments at a constant reservoir ionic strength. Data from these uncontrolled experiments were used to determine rates of supersaturation, induction times, and the number and size of the resultant lysozyme crystals. Control experiments were performed by introducing step changes in the reservoir ionic strength. The step changes were initiated by comparing in situ rates of supersaturation with the rates of supersaturation calculated from the uncontrolled data. Monitoring the concentration changes of the lysozyme within the hanging drop permits a measurement of the level of supersaturation of the system and enhances the possibility of dynamic control of the crystallization process.

The use of Raman spectroscopy for in situ monitoring of lysozyme concentration during crystallization in a hanging drop

Fiber optic Raman spectroscopy combined with a partial least-squares regression model was investigated as a means to monitor lysozyme concentration during crystallization in a hanging drop experiment in real time. Raman spectral features of the buffer and protein were employed to build the regression model. This model was used to calculate the compositional changes within the hanging drop. The use of fibre optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous media, results in a powerful noninvasive probe of the changing environment within the solution. These preliminary findings indicate that solubility as well as supersaturation measurements can be made.

Purification process for succinic acid produced by fermentation

Succinic acid is a versatile four-carbon dicarboxylic acid. It can be used commerically as an intermediate chemical for the manufacture of 1,4-butanediol, maleic anhydride, and many other chemicals. Succinic acid can be produced by the fermentation of carbohydrates. A complete process for the production and purification of succinic acid from carbohydrates has been developed. The process includes fermentation, desalting electrodialysis, water-splitting electrodialysis, and crystallization to produce a pure crystalline succinic acid. This article will present experimental work performed in the development of this process.

Production of the bioactive compound eritadenine by submerged cultivation of shiitake (Lentinus edodes) mycelia.

Fruit bodies and mycelia of shiitake mushroom ( Lentinus edodes) have been shown to contain the cholesterol-reducing compound eritadenine, 2( R),3( R)-dihydroxy-4-(9-adenyl)butyric acid. In the search for a production method for eritadenine, shiitake mycelia were investigated in the present study. The mycelia were cultivated both in shake flasks and in bioreactors, to investigate the effects of pH, stirring rate, and reactor type on the production and distribution of eritadenine. Both the biomass and the culture broth were examined for their eritadenine content. In the shake flasks, the final concentration of eritadenine was 1.76 mg/L and eritadenine was equally distributed between the mycelia and the growth media. In the bioreactors, the shiitake mycelia were found to contain eritadenine in relatively low levels, whereas the majority, 90.6-98.9%, was detected in the growth media. Applying a stirring rate of 250 rpm during bioreactor cultivation resulted in the highest eritadenine concentrations: 10.23 mg/L when the pH was uncontrolled and 9.59 mg/L when the pH was controlled at 5.7. Reducing the stirring rate to 50 rpm resulted in a decreased eritadenine concentration, both at pH 5.7 (5.25 mg/L) and when pH was not controlled (5.50 mg/L). The mycelia in the shake flask cultures appeared as macroscopic aggregates, whereas mycelia cultivated in bioreactors grew more as freely dispersed filaments. This study demonstrates for the first time the extra- and intracellular distribution of eritadenine produced by shiitake mycelial culture and the influence of reactor conditions on the mycelial morphology and eritadenine concentrations.