Substrate oscillations boost recombinant protein release from Escherichia coli.

Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60 %, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4 min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45 % of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies.

Effects of temperature shifts and oscillations on recombinant protein production expressed in Escherichia coli.

Escherichia coli is widely used host for the intracellular expression of many proteins. However, in some cases also secretion of protein from periplasm was observed. Improvement of both intracellular and extracellular production of recombinant protein in E. coli is an attractive goal in order to reduce production cost and increase process efficiency and economics. Since heat shock proteins in E. coli were reported to be helpful for protein refolding and hindering aggregation, in this work different types of single and periodic heat shocks were tested on lab scale to enhance intracellular and extracellular protein production. A single heat shock prior to induction and different oscillatory temperature variations during the induction phase were executed. The results showed that these variations influence protein production negatively. In other words, 45 and 50 % reduction in extracellular protein production were observed for the single heat shock and oscillated temperature between 35 and 40 °C, respectively. However, the oscillatory temperature approach introduced in this study is recommended as a tool to quantitatively analyze the effects of inhomogeneous temperature on cell physiology and productivity in large-scale bioreactors.

Sustainable bioconversion of potato peel wastes into ethanol and biogas using organosolv pretreatment.

Potato processing industries generate considerable amounts of residues, i.e., potato peel wastes (PPW). Valorization of PPW for bioethanol and biogas production via a biorefining process was investigated in this study. Organosolv pretreatment was performed on the PPW using 50-75% (v/v) ethanol solution at 120-180 °C with/without the presence of 1% (w/w) H2SO4 (as a catalyst). After the pretreatment, the solvent, i.e., ethanol, was recovered by distillation. Catalyzed organosolv pretreatment using 50% (v/v) ethanol at 120 °C followed by enzymatic hydrolysis resulted in a high hydrolysate yield of 539.8 g glucose/kg dry PPW that was successfully fermented to 224.2 g ethanol/kg dry PPW. To recover more energy, the liquid fraction of the pretreatment remained after solvent recovery and the unhydrolyzed solids that remained from the enzymatic hydrolysis were anaerobically digested. From each kg of dry PPW, the anaerobic digestion produced 57.9 L biomethane. Thus, the biorefinery comprising ethanolic organosolv pretreatment, solvent recovery, enzymatic hydrolysis, ethanolic fermentation, and anaerobic digestion of residues was produced 8112 kJ energy per kg of dry PPW.

Waste Gastro-intestinal Wall of Sheep as an Alternative Nutrition Source for Cultivation of Dunaliella salina.

In the production of natural sausage casings, three layers of serosa, muscular, and mucosa are removed from gastro-intestinal wall of sheep as waste materials. The submocusa layer is taken for further processing. There is no report about generating added value out of these wastes. In this study, a novel approach was introduced for bioconversion of waste gastro-intestinal wall (WGW) to a value-added product. Alkaline hydrolysis of WGW was investigated and the hydrolysate was utilized for cultivation of Dunaliella salina, a value-added biomass. The hydrolysate that contained the highest total soluble protein was used for three sets of cultivations on different medium compositions, i.e., (1) cultivations on the modified Johnson's medium enriched with different percentage of hydrolysate (0.5, 1, 2.5, 5, and 10 (%v/v)), (2) cultivations on modified Johnson's medium which was free of nitrogen and carbon sources and enriched with different percentage of hydrolysate (0.5, 1, 2.5, 5, and 10 (%v/v), and (3) cultivation on modified Johnson's medium which was free of nitrogen source and enriched with 2.5% hydrolysate. The results showed that WGW contained 60.7, 8.4, 15.8, and 15.2% protein, lipid, moisture, and ash, respectively and the enrichment of the medium with the hydrolysate (2.5%) increased biomass productivity by 20%. Additionally, substitution of 2.5% hydrolysate for nitrogen source (KNO3) resulted in the same biomass productivity. The results of this study revealed the potential of the hydrolysate as an alternative for KNO3 in cultivation of D. salina. Overall, this work proposed a novel approach for converting waste gastro-intestinal wall to value.

Efficient superantioxidant and biofuel production from microalga Haematococcus pluvialis via a biorefinery approach.

A biorefinery approach was implemented to produce a superantixoident, i.e., astaxanthin, and biofuels, i.e., ethanol and biogas, from the biomass of microalga Haematococcus pluvialis. The hydrolysis of residual biomass obtained from astaxanthin extraction was conducted using α-amylase and glucoamylase for hydrolysis of α-glucans and a mixture of cellulases for ß-glucan hydrolysis. Four different hyudrolysis processes were employed and the efficiency of 97.2% over the total residual glucan was obtained, which was then fermented to produce 0.21 g ethanol/g residual biomass. The residuals obtained from astaxanthin extraction and fermentation were anaerobically digested to produce biomethane. The yield of biomethane was 264.8 ml/g volatile solids, 2.9 fold greater than methane yield from raw microalgal biomass. Overall, the process of astaxanthin extraction and consecutive production of ethanol and biogas from H. pluvialis biomass was recognized as a promising process to produce 45.8 g astaxanthin and 7095.3 KJ energy per Kg of raw biomass.

Investigation of the physiological response to oxygen limited process conditions of Pichia pastoris Mut(+) strain using a two-compartment scale-down system.

Inhomogeneities in production-scale bioreactors influence microbial growth and product quality due to insufficient mixing and mass transfer. For this reason, lots of efforts are being made to investigate the effects of gradients that impose stress in large-scale reactors in laboratory scale. We have implemented a scale-down model which allows separating a homogeneous part, a stirred tank reactor (STR), and a plug flow reactor (PFR) which mimics the inhomogeneous regimes of the large-scale fermenters. This scale-down model shows solutions to trigger oxygen limited conditions in the PFR part of the scale-down setup for physiological analysis. The goal of the study was to investigate the scale-up relevant physiological responses of Pichia pastoris strain to oxygen limited process conditions in the above mentioned two-compartment bioreactor setup. Experimental results with non-induced cultures show that the specific growth rate significantly decreased with increasing the exposure time to oxygen limitation. In parallel more by-products were produced. Examining physiological scalable key parameters, multivariate data analyses solely using on-line data revealed that different exposures to the oxygen limitation significantly affected the culture performance. This work with the small scale-downs setup reflects new approaches for a valuable process development tool for accelerating strain characterization or for verifying CFD simulations of large-scale bioreactors. As a novel methodological achievement, the combination of the two-compartment scale-down system with the proposed multivariate techniques of solely using on-line data is a valuable tool for recognition of stress effects on the culture performance for physiological bioprocess scale-up issues.

Effect of post-induction substrate oscillation on recombinant alkaline phosphatase production expressed in Escherichia coli.

Microorganisms are exposed to fast changes in microenvironment in large scale bioreactors. Because of their fast response to the changes, overall performance of biological system in small scale differs from large scale. Hence the variations in the environment that microorganisms are living in are mimicked in small scale. For this purpose one way is to feed substrate into the bioreactor in an oscillatory profile. In this work two different types of triangular oscillatory feeding profiles were applied as the post induction feeding strategy in intracellular recombinant alkaline phosphatase production expressed in Escherichia coli to find out if this biological system behaves in inhomogeneous environment differently. On line and offline measurements provide evaluation of product quality and quantity. Then the results of the experiments were compared with those of the control run at which constant feeding rate was executed. The results showed that oscillatory feeding at which cells were not starved led to higher yield of protein per substrate (0.027C-mol/C-mol) and higher activity per protein (0.79U/mg) when compared to a constant feeding rate (0.011C-mol/C-mol and 0.11U/mg).