Fed-batch Technique Research Articles

Production of a cellulose-aminating polysaccharide from a filamentous sulfur-oxidizing bacterium, Thiothrix nivea, grown lithotrophically or mixotrophically.

Glucosaminoglucan (β-1,4-linked glucose and glucosamine) produced by a mixotrophic sulfur-oxidizing bacterium, Thiothrix nivea, is a useful cellulose-aminating agent. Lithotrophic and mixotrophic glucosaminoglucan production were examined using fed-batch techniques. A jar fermenter was used for the fed-batch cultivation. Glucosaminoglucan was extracted from T. nivea using diluted HCl. Lithotrophic growth was detected by feeding with Na2S as the energy source, and 12mg L-culture-1 of glucosaminoglucan was obtained. In contrast, no growth was observed with Na2S2O3. Similarly, mixotrophic growth in the presence of acetic acid was promoted by Na2S, whereas Na2S2O3 had no effect. When acetic acid and Na2S were added, 470mg L-culture-1 of glucosaminoglucan was obtained. T. nivea was cultured and glucosaminoglucan was produced lithotrophically using Na2S for feeding. Na2S is also indispensable for mixotrophic growth and glucosaminoglucan production, indicating that sulfide oxidation pathways control the TCA cycle. The involvement of the SOX pathway (for thiosulfate oxidation) in the activation of energy metabolism is doubtful because neither lithotrophic nor mixotrophic growth was promoted by Na2S2O3. Based on these results, we assumed that T. nivea is facultatively mixotrophic (lithotrophic growth is possible in addition to organotrophic growth in the presence of sulfide (Na2S)), rather than obligately mixotrophic.

Comparison of Growth of Chlorella vulgaris in Flat-Plate Photobioreactor Using Batch, Fed-Batch, and Repeated Fed-Batch Techniques with Various Concentrations of Walne Medium

Chlorella vulgaris was autotrophically grown in a Flat-Plate Photobioreactor (FPPB) using batch (BC), fed-batch (FB), and repeated fed-batch (RPF) cultivations. BC stage was done in the 20 L of working volume. During FB stages, 20 L of fresh medium was added to reach the final volume of 60 L. In the RPF stages, 20 L of broth was harvested, and then a similar volume of fresh medium was added to keep the working volume. This study aimed to evaluate the effect of Walne media (with various concentrations of composition) and cultural techniques on algal growth. Three different concentrations of Walne medium (original [coded as ´1], double [coded as ´2], and triple [coded as ´3] in concentrations of the original medium) was used for cultivations. Not only culture techniques but also various media affected biomass and chlorophyll productivity. RPF (with ´2 cultivation) gave the highest biomass productivity. However, RPF (with ´1 cultivation) gave the highest chlorophyll productivity. So, this study showed much fundamental information for the industrial production of Chlorella vulgaris biomass using flat-plate photobioreactors.

Dynamic multiscale metabolic network modeling of Chinese hamster ovary cell metabolism integrating N-linked glycosylation in industrial biopharmaceutical manufacturing.

Experimental and modeling work, described in this article, is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody protein production. CHO cells are one of the primary hosts for monoclonal antibodies production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed-batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and CO2 content, and dissolved oxygen. A total of 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N-glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional eight reactions are considered for accumulation of the N-glycosylation products in the extracellular environment and one reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions, and membrane transport. With the applied modeling procedure, different operational scenarios and fed-batch techniques can be tested.

Biocatalytic production of compound K in a deep eutectic solvent based on choline chloride using a substrate fed-batch strategy

This study involved the development of a β-glucosidase-catalyzed hydrolysis method based on a deep eutectic solvent (DES), choline chloride-ethylene glycol 2:1, and continuous feed technique to overcome the difficulty of high-concentration ginsenoside hydrolysis. A productivity of 142 mg·L−1·h−1 was achieved with the following conditions: 30 vol% DES, pH 5.0, 55 °C, and substrate concentration of 12 mM. In the presence of DES, the affinity and catalytic efficiency of β-glucosidase to Rd increased by 49 and 64%, respectively, which promoted the continuation of hydrolysis. Moreover, conformation of β-glucosidase was mostly retained, as confirmed by spectral information. Through a combination of a substrate fed-batch technique to reduce the inhibitory effects of substrates and products, the CK conversion rate increased by 44% compared to traditional single-batch in pure buffer. This report describes a practical method for the continuous conversion of natural compounds through biological processes and solvent engineering.

A novel approach using low-cost Citrus limetta waste for mixotrophic cultivation of oleaginous microalgae to augment automotive quality biodiesel production

The present study reports the use of Citrus limetta (CL) residue for cultivating Chlorella sp. mixotrophically to augment production of biodiesel. The cultivation of Chlorella sp. using CL as media was carried out by employing a fed-batch technique in open tray (open tray+CL) and in software (BioXpert V2)-attached automated photobioreactor (PBR+CL) systems. Data showed the limit of nitrogen substituent and satisfactory organic source of carbon (OSC) in CL, causing > 2-fold higher lipid content in cells, cultivated in both the systems than in control. For the cells grown in both the systems, ≥ 3-fold enhancement in lipid productivity was observed than in control. The total fatty acid methyl ester (FAME) concentrations from lipids extracted from cells grew in PBR+CL and in open tray+CL techniques were calculated as 50.59% and 38.31%, respectively. The PBR+CL system showed improved outcomes for lipid content, lipid and biomass productivity, FAME characteristics and physical property parameters of biodiesel than those obtained from the open tray+CL system. The physical property parameters of biodiesel produced from algal cells grown in PBR+CL were comparable to existing fuel standards. The results have shown lower cold filter plugging point (- 6.57 °C), higher cetane number (58.04) and average oxidative stability (3.60 h). Collectively, this investigation unveils the novel deployment of CL as a cost-effective feedstock for commercialisation of biodiesel production.

The substratostat - an automated near-infrared spectroscopy-based variable-feed system for the continuous alcoholic fed-batch fermentation of high gravity grape must at static sugar concentrations

Aim: High sugar concentrations in musts cause a hyperosmotic stress response in Saccharomyces cerevisiae increasing the risk of sluggish and stuck alcoholic fermentations and/or causing high acetic acid levels. Applying a fed-batch technique where sugar levels are kept at a constant, low rate throughout fermentations reduces this stress but requires in-situ quantification of sugars and process automation for practicability. The aim of this work was to develop and validate a near-infrared (NIR) spectroscopy method allowing for the continuous in-situ quantification of total fermentable sugars in fully turbid alcoholic fermentations of grape musts. Calibration models for glucose, fructose and the fermentation product ethanol were also established.Methods and results: A research Fourier-transform NIR spectrophotometer equipped with a transflectance probe was used to acquire spectra from 240 natural and semisynthetic standards from fermentations conducted using varying concentrations of yeast and yeast nutrients. Using chemometric software, calibration models for total sugars, glucose, fructose and ethanol demonstrated R2 values >0.93 and prediction error (RMSEP) values of 11.6 g l-1, 12.3 g l-1, 10.2 g l-1, and 0.328 % v/v, respectively. The method was integrated with modern process automation technology and was able to maintain sugar concentrations within 5 g l-1 of the 45 g l-1 setpoint adjusted during alcoholic fermentations.Conclusions: The NIR calibration models generated allow prediction of total sugar levels accurately enough to conduct fully automated fed-batch grape must fermentations at constant substrate concentrations. Application of a transflectance probe measuring a high proportion of back-scattered radiation proved useful and necessary considering the high degree of turbidity during fermentations. Placement of the measurement probe in a recirculation loop decreased interference from biomass sedimentation and adherence of CO2 bubbles.Significance and impact of the study: This study presents a fully automated system to carry out fed-batch fermentations which allow circumventing the hyperosmotic stress response of S. cerevisiae during alcoholic fermentations. Calibrated for other substrates, the system may be used in other food and non-food fermentations, too.

Metabolism of Schizosaccharomyces pombe under reduced osmotic stress conditions afforded by fed-batch alcoholic fermentation of white grape must

Strains of Schizosaccharomyces pombe are being increasingly investigated with regards to their grape winemaking potential either in combination with the typical production yeast, Saccharomyces cerevisiae, or in monoseptic fermentations. Their ethanol tolerance and ability to degrade L-malic acid is oenologically convenient but contrasts with the comparatively high acetic acid and acetaldehyde formation potential which is considered undesirable, especially in white winemaking. The purpose of this work was to investigate the performance of a selected S. pombe strain in monoseptic femerntations of white grape must. Traditional batch fermentations were compared with an innovative and automated fed-batch fermentation technique were sugar concentrations are kept low during fermentations to decrease sugar induced osmotic stress. Because of its known effect on growth and ethanol tolerance, the effect of Mg was also tested. While Mg supplementation was not shown to significantly influence residual values of sugars, ethanol, glycerol, organic acids and acetaldehyde, the application of the fed-batch technique led to a fundamental change in yeast physiology. While glycerol values were only slightly reduced, the fed-batch approach allowed obtaining wines devoid of acetic acid whose levels were considerable in wines produced by the traditional batch technique (0.6 g/L). The work demonstrates that the acetic acid metabolism of S. pombe is associated to sugar induced osmotic stress such as for S. cerevisiae, too, and may be controlled by application of suitable fermentation techniques for winemaking.

Development of fed-batch process for high-yielding β-glucosidase displayed on cell surface of industrial yeast Saccharomyces cerevisiae

Here, we developed a fed-batch process for the cultivation of multistress tolerant Saccharomyces cerevisiae engineered to display β-glucosidase (BGL) on the cell surface. Although cell growth was promoted by increasing the amount of oxygen supplied, increasing the overall volumetric oxygen transfer coefficient rather decreased the specific BGL activity of yeast cells probably because of the high agitation speed used. To suppress the formation of ethanol, the method of feeding glucose was investigated. Namely, the timely addition of glucose and glucose feed rate were found crucial for increasing both cell concentration and specific BGL activity. When the lowest concentration of dissolved oxygen at 9h was used as an indicator, the lowest glucose feed rate of 1g/(L·h) provided the maximum BGL activity of 673.6U/g-dry cell, which is 1.6-fold higher than that in batch cultivation. By employing the fed-batch technique together with the improved gene cassette containing the SED1 promoter and anchoring domain, we found that the cell concentration and specific BGL activity continued to increase, reaching 15.4 g-dry cell/L and 1866.6U/g-dry cell after 48h, respectively. The results show the advantage of the fed-batch process for realizing the potential of cell-surface display systems in S. cerevisiae.

Fed-batch fermentation of nipa sap to acetic acid by Moorella thermoacetica (f. Clostridium thermoaceticum)

An efficient process for conversion of nipa sap to acetic acid was developed. Nipa sap was hydrolyzed with invertase and provided glucose as well as fructose as main sugars. Batch fermentation of glucose and fructose was inadequate with increased substrate concentration. By contrast, fed-batch technique on hydrolyzed nipa sap with high feeding rate drastically increased acetic acid concentration and productivity to be 42.6 g/L and 0.18 g/(L/h), respectively. All the sugars in hydrolyzed nipa sap were consumed, with acetic acid yield of 0.87 g/g sugar. Overall, nipa sap as hydrolyzed with invertase was efficiently fermented to acetic acid, which is a valuable chemical and a potential biorefinery intermediate.

Environmentally Friendly Production of D(-) Lactic Acid by Sporolactobacillus nakayamae: Investigation of Fermentation Parameters and Fed-Batch Strategies.

The interest in the production of lactic acid has increased due to its wide range of applications. In the present study, the variables that affect fermentative D(−) lactic acid production were investigated: neutralizing agents, pH, temperature, inoculum percentage, agitation, and concentration of the medium components. An experimental design was applied to determine the optimal concentrations of the medium components and fermentation was studied using different feeding strategies. High production (122.41 g/L) and productivity (3.65 g/L·h) were efficiently achieved by Sporolactobacillus nakayamae in 54 h using a multipulse fed-batch technique with an initial medium containing 35 g/L of yeast extract (byproduct of alcohol production), 60 g/L of crystallized sugar, and 7.5 mL/L of salts. The fermentation process was conducted at 35°C and pH 6.0 controlled by NaOH with a 20% volume of inoculum and agitation at 125 rpm. The production of a high optically pure concentration of D(−) lactic acid combined with an environmentally friendly NaOH-based process demonstrates that S. nakayamae is a promising strain for D(−) lactic acid production.

In vitro growth of Curcuma longa L. in response to five mineral elements and plant density in fed-batch culture systems.

Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.

Recombinant Erwinia carotovora l-asparaginase II production in Escherichia coli fed-batch cultures

Abstract - Asparaginases are the cornerstone therapy of many successful combination regimens for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. The aim of this work was to produce recombinant Erwinia carotovora L-asparaginase II in Escherichia coli fed-batch cultures. Using a robust fed-batch technique with pre-determined exponential feeding rates, our bioreactor culture system yielded 30.7 grams of dry cell weight and 0.9 grams of soluble rErAII protein per liter of culture broth. The homogeneous rErAII activity was determined by isothermal titration calorimetry (ITC). The enzyme K m values for the main substrates L-Asn and L-Gln were 33x10 -6 M and 10x10 -3 M, respectively. Our work shows that it is possible to produce an active homogeneous rErAII enzyme in the soluble cell fraction through IPTG-induced E. coli fed-batch cultivation. Keywords : Acute lymphoblastic leukemia; Asparaginase; Erwinia carotovora ; Bioreactor; Isothermal titration calorimetry;

Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

BackgroundBioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.ResultsHere, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.ConclusionsCultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.

Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

BackgroundThe prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.ResultsA synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.ConclusionsA simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

Biodegradation of toluene at high initial concentration in an organic–aqueous phase bioprocess with nitrate respiration

An aerobic organic–aqueous system with forced aeration was shown to be inefficient in preventing significant volatile aromatic compounds loss in gassed systems since air sparging in n-hexadecane under abiotic conditions could reduce the toluene concentration from 2.1 g/L to about 0.5 g/L in 3 days with a gassing rate of 1VVM at 20 °C. However, the presence of such an organic phase was found to significantly reduce substrate loss in aerobic conditions in comparison to pure aqueous systems. It was thus decided to develop a new bioprocess based on an anaerobic microbial system operated in an organic–aqueous phase with nitrate respiration. The denitrifying bacterium used, Thauera aromatica K172, was produced by cultivation on sodium benzoate as carbon source under anaerobic conditions. This cultivated biomass (1.5 g/L) was shown to retain its ability to efficiently metabolize toluene in a biphasic medium without any significant loss of organic compound in the gas phase. Toluene biodegradation was thus performed in a biphasic system using a fed-batch technique involving sequential adding of both toluene and nitrate. The reaction rate with an initial concentration of toluene close to 14.5 g/L in hexadecane was found to be close to 0.5 g/L day and the molar stoichiometry of solute metabolization to nitrate reduction was close to 1:6. This work demonstrated that the denitrifying bacteria could efficiently degrade toluene in hexadecane–aqueous phase systems in which toxic compound release in the environment was prevented.

Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

BackgroundHepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus.ResultsLarge-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth.ConclusionIn comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.

Challenges in production of recombinant proteins

The sequencing of the human genome and genomes from lower species has led molecular biology research into the era of proteomics. Starting from the cDNA’s, a large number of proteins have to be expressed in quantities large enough to allow determining of their structure and function, which forms the basis for the development of new pharmaceutical products. The larger number of these proteins is most probably new to us, and furthermore, we expect that they will involve a high-degree of complexity. The process development relies on ‘‘trial-and-error’’ methods to provide the bases for the choice of production method since we still do not have the knowledge that could guide this choice from the cDNA structure alone. We must therefore expect that the number and complexity of the proteins will require an equally wide portfolio of production methods. For this purpose, there is thus shown a large interest in new production systems, which could be used for a multitude of proteins as well as for a unique protein. Proteins for crystallisation and functional studies must be structurally correct and produced to a high degree of purity. In the valuation of recombinant systems for the production of proteins, the emphasis is therefore also placed on product quality. This issue of Bioprocess and Biosystems Engineering includes a summary of the research on a new production system based on cells from a moss. The system uses a strain, which has been genetically engineered to produce products with strictly human-specific glycosylation patterns (Decker and Reski), which gives great process advantages compared with the use of e.g., yeasts and animal cell systems. One might imagine that many years of research in bioprocess development would have led also to the development of general tools which could be used in the context of production for structural genomics too; but in many cases, these tools involve technologies which are not applicable on a small scale, since they are based on operator-supervised control systems of a technical complexity which cannot be used in a micro-scale, multi-parallel cultivation format. An example of such a very powerful control system is the fed-batch technique. This is the only cultivation technique, which allows the accumulation of cells to a high cell density. But in addition, this technique leads further to the establishment of different growth rates, which has been shown to be an important factor for the control of both productivity and product quality, particularly regarding the solubility of a protein. To achieve this on a small scale, a technology is proposed in this issue (Backlund et al.), where cell engineering is used to enable the control of substrate limitation at cell rather than at reactor level. There are great expectations that the large investments in time and money in the genome and proteome projects will lead to an increased number of new and better recombinant bioproducts. In those cases where these products are intended for use as biopharmaceuticals, great demands are made not only on the quality of the product but also on the reproducibility of the processes. This increases the demand for powerful analytical techniques capable of resolving quality steering issues very early on in the process as well as the appropriate control technology. Control algorithms are being developed in many lines of business outside the pharmaceutical sector, and the relatively weak development on the pharmaceutical G. Larsson (&) School of Biotechnology, Royal Institute of Technology, Roslagstullsbacken 21, Stockholm SE10691, Sweden e-mail: gen@biotech.kth.se

Bioreactor control using a probing feeding strategy and mid-ranging control

The paper presents a fed-batch fermentation technique for bioreactors operating close to their maximum oxygen transfer capacity. The method combines the advantages of the probing feeding strategy and the temperature limited fed-batch technique. When the maximum oxygen transfer capacity of the reactor is reached, the temperature is decreased to lower the oxygen demand. To achieve a good control of the dissolved oxygen a mid-ranging controller manipulating the stirrer speed and the temperature is used. The feeding strategy is analysed and it is also illustrated by simulations and an experiment.

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l−1 of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l−1 and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h−1, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l−1 h−1, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l−1, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l−1 h−1. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l−1 and 1.345 g l−1 h−1, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.

Total amino acid stabilization during cell-free protein synthesis reactions

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.