Modeling and Optimization of Gas Sparging-Assisted Bacterial Cultivation Broth Microfiltration by Response Surface Methodology and Genetic Algorithm

Production of highly efficient biomass-based microbial biopesticides significantly depends on downstream processing in terms of obtaining as high concentration of viable cells as possible. Microfiltration is one of the recommended operations for microbial biomass separation, but its main limitation is permeate flux decrease due to the membrane fouling. The effect of air sparging as a hydrodynamic technique for improvement of permeate flux during microfiltration of Bacillus velezensis cultivation broth was investigated. Modeling of the microfiltration was performed using the response surface methodology, while desirability function approach and genetic algorithm were applied for optimization, i.e., maximization of permeate flux and minimization of specific energy consumption. The results have revealed antagonistic relationship between the investigated dependent variables. The optimized values of superficial feed velocity and transmembrane pressure were close to the mean values of the investigated value ranges (0.68 bar and 0.96 m/s, respectively), while the optimized value of superficial air velocity had a more narrow distribution around 0.25 m/s. The results of this study have revealed a significant improvement of microfiltration performance by applying air sparging, thus this flux improvement method should be further investigated in downstream processing of different bacterial cultivation broths.

Exploring functionality of the reverse β-oxidation pathway in Corynebacterium glutamicum for production of adipic acid

Background Adipic acid, a six-carbon platform chemical mainly used in nylon production, can be produced via reverse β-oxidation in microbial systems. The advantages posed by Corynebacterium glutamicum as a model cell factory for implementing the pathway include: (1) availability of genetic tools, (2) excretion of succinate and acetate when the TCA cycle becomes overflown, (3) initiation of biosynthesis with succinyl-CoA and acetyl-CoA, and (4) established succinic acid production. Here, we implemented the reverse β-oxidation pathway in C. glutamicum and assessed its functionality for adipic acid biosynthesis. Results To obtain a non-decarboxylative condensation product of acetyl-CoA and succinyl-CoA, and to subsequently remove CoA from the condensation product, we introduced heterologous 3-oxoadipyl-CoA thiolase and acyl-CoA thioesterase into C. glutamicum. No 3-oxoadipic acid could be detected in the cultivation broth, possibly due to its endogenous catabolism. To successfully biosynthesize and secrete 3-hydroxyadipic acid, 3-hydroxyadipyl-CoA dehydrogenase was introduced. Addition of 2,3-dehydroadipyl-CoA hydratase led to biosynthesis and excretion of trans-2-hexenedioic acid. Finally, trans-2-enoyl-CoA reductase was inserted to yield 37 µg/L of adipic acid. Conclusions In the present study, we engineered the reverse β-oxidation pathway in C. glutamicum and assessed its potential for producing adipic acid from glucose as starting material. The presence of adipic acid, albeit small amount, in the cultivation broth indicated that the synthetic genes were expressed and functional. Moreover, 2,3-dehydroadipyl-CoA hydratase and β-ketoadipyl-CoA thiolase were determined as potential target for further improvement of the pathway.

Effect of different carbon and nitrogen sources combination in medium for production of biocontrol agent Trichoderma harzianum

The increasing usage of chemicals for plant protection in recent years has become a serious problem. One of the possible solutions is use of beneficial microorganisms instead of synthetic fungicides, which will contribute to the protection of the environment and human health. Since the fungi Aspergillus flavus and Fusarium graminearum are the most important pathogens that cause maize diseases and produce mycotoxins, the potential of Trichoderma harzianum for biocontrol of both phytopathogens was examined in this paper. The aim of this paper was to study the influence of different carbon and nitrogen combinations in the medium for T. harzianum production. T. harzianum was cultivated in Erlenmeyer flasks and the effect of cultivation broth against selected maize pathogens was tested using well diffusion method. The results of this study showed that the combination of different carbon and nitrogen sources in the T. harzianum cultivation medium statistically significantly affects the production of Trichoderma cultivation broth effective on two tested phytopathogens. Dextrose as a carbon source and soybean flour as a nitrogen source proved to be the best combination in the medium for production of T. harzianum cultivation broth effective on A. flavus and F. graminearum. Maximal inhibition zone diameters of 31 mm and 56.33 mm were registered in those medium formulations for A. flavus and F. graminearum, respectively. These researches represent an important step for further research in which a medium of low market value would be selected. This would reduce the price of the production process but also the final product.

Dynamic Modeling Using Artificial Neural Network of Bacillus Velezensis Broth Cross-Flow Microfiltration Enhanced by Air-Sparging and Turbulence Promoter

Cross-flow microfiltration is a broadly accepted technique for separation of microbial biomass after the cultivation process. However, membrane fouling emerges as the main problem affecting permeate flux decline and separation process efficiency. Hydrodynamic methods, such as turbulence promoters and air sparging, were tested to improve permeate flux during microfiltration. In this study, a non-recurrent feed-forward artificial neural network (ANN) with one hidden layer was examined as a tool for microfiltration modeling using Bacillus velezensis cultivation broth as the feed mixture, while the Kenics static mixer and two-phase flow, as well as their combination, were used to improve permeate flux in microfiltration experiments. The results of this study have confirmed successful application of the ANN model for prediction of permeate flux during microfiltration of Bacillus velezensis cultivation broth with a coefficient of determination of 99.23% and absolute relative error less than 20% for over 95% of the predicted data. The optimal ANN topology was 5-13-1, trained by the Levenberg–Marquardt training algorithm and with hyperbolic sigmoid transfer function between the input and the hidden layer.

Marine Fungus Aspergillus chevalieri TM2-S6 Extract Protects Skin Fibroblasts from Oxidative Stress

The strain Aspergillus chevalieri TM2-S6 was isolated from the sponge Axinella and identified according to internal transcribed spacer (ITS) molecular sequence homology with Aspergillus species from the section Restricti. The strain was cultivated 9 days on potato dextrose broth (PDB), and the medium evaluated as antioxidant on primary normal human dermal fibroblasts (NHDF). The cultivation broth was submitted to sterile filtration, lyophilized and used without any further processing to give the Aspergillus chevalieri TM2-S6 cultivation broth ingredient named ACBB. ACCB contains two main compounds: tetrahydroauroglaucin and flavoglaucin. Under oxidative stress, ACCB showed a significant promotion of cell viability. To elucidate the mechanism of action, the impact on a panel of hundreds of genes involved in fibroblast physiology was evaluated. Thus, ACCB stimulates cell proliferation (VEGFA, TGFB3), antioxidant response (GPX1, SOD1, NRF2), and extracellular matrix organization (COL1A1, COL3A1, CD44, MMP14). ACCD also reduced aging (SIRT1, SIRT2, FOXO3). These findings indicate that Aspergillus chevalieri TM2-S6 cultivation broth exhibits significant in vitro skin protection of human fibroblasts under oxidative stress, making it a potential cosmetic ingredient.

Integrated Clarification and Purification of Monoclonal Antibodies by Membrane Based Separation of Aqueous Two-Phase Systems

Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.

Utilization of maltose enriched spent grains liquor for xanthan production

The present study examines xanthan production by Xanthomonas campestris under aerobic conditions on spent grains liquor based cultivation media containing four different amounts of maltose. All four experiments were performed simultaneously, so that all stages of the biotechnological process would be carried out under identical conditions. In addition to the cultivation parameters (pH value, contents of dissolved oxygen, reducing sugars and assimilable nitrogen, turbidity and trickling time), xanthan yield and sugar conversion percentage were also determined. Based on the results of the experiments maltose addition up to 5% to spent grains liquor does not hinder the cultivation course. On the other hand, the conversion of sugars into xanthan decreases with the increase of maltose content to 5% and a large amount of sugars lags in the cultivation broth.