Extractive Fermentation Employing Ion-Exchange Resin to Enhance Cell Growth and Production of Metabolites Subject to Product or By-Product Inhibition

In recent years, commercial production of proteins and metabolites from microbial fermentation for industrial applications has increased significantly. Innovative approaches are directed towards the improvement of the conventional batch fermentation method and the segregated downstream processing of target product to improve the overall process efficiency and to ensure that the process is economically viable. Feedback inhibition is a common problem faced during fermentation process when the concentration of end-product/by-product reaches a certain level. The excessive accumulation of end-product/by-product in the culture may inhibit the growth of cell and represses the secretion of target metabolite. In the production of many fermentative products such as antibiotics, amino acids, and fungal metabolites, a serious problem of feedback inhibition is often encountered. Cultivation of lactic acid bacteria and recombinant bacteria is usually subjected to by-product inhibition. Hence, extractive fermentation via in situ ion-exchange-based adsorptive technique is a possible approach to be used industrially to mitigate feedback inhibition, aimed at enhancing fermentation performance. In this chapter, advances in this area were presented. Strategies to overcome problem related to product/by-product inhibitions by this technique via dispersed, external, and internal resin system, and the general methodology in the implementation of the technique were also discussed

Growth Enhancement of Probiotic Pediococcus acidilactici by Extractive Fermentation of Lactic Acid Exploiting Anion-Exchange Resin

Fermentation employing lactic acid bacteria (LAB) often suffers end-product inhibition which reduces the cell growth rate and the production of metabolite. The utility of adsorbent resins for in situ lactic acid removal to enhance the cultivation performance of probiotic, Pediococcus acidilactici was studied. Weak base anion-exchange resin, Amberlite IRA 67 gave the highest maximum uptake capacity of lactic acid based on Langmuir adsorption isotherm (0.996 g lactic acid/g wet resin) compared to the other tested anion-exchange resins (Amberlite IRA 410, Amberlite IRA 400, Duolite A7 and Bowex MSA). The application of Amberlite IRA 67 improved the growth of P. acidilactici about 67 times compared to the control fermentation without resin addition. Nevertheless, the in situ addition of dispersed resin in the culture created shear stress by resins collision and caused direct shear force to the cells. The growth of P. acidilactici in the integrated bioreactor-internal column system containing anion-exchange resin was further improved by 1.4 times over that obtained in the bioreactor containing dispersed resin. The improvement of the P. acidilactici growth indicated that extractive fermentation using solid phase is an effective approach for reducing by-product inhibition and increasing product titer

Molybdenum reductase in Enterobacter cloacae

Under anaerobic conditions in glucose–yeast extract medium with phosphate, Enterobacter cloacae strain 48 grew well and reduced Mo6+, to Mo5+. The activity of Mo6+-reductase was measured by the formation of molybdenum blue (complexation between Mo5+ and phosphate ion). Models based on logistic and Luedeking–Piret equations were found adequate to describe the growth of E. cloacae and Mo6+-reductase production. Mo6+-reductase production was found to be a growth-associated process. Washed intact cells, membrane fraction (after disruption using a sonicator) and fluid supernatant (after cell disruption) were able to reduce Mo6+. However, Mo6+-reductase activity was much lower in the supernatant fluid. The (NH4)2SO4-precipitated Mo6+-reductase extract from fluid supernatant was assayed for its properties. The optimum pH and temperature for Mo6+-reductase activity were 8 and 30°C, respectively. The apparent Michaelis–Menten constant (Km) and a maximum velocity (Vmax) were 16.5mm and 0.0192μmol/ml.h, respectively

Assessment of molecular recognition element for the quantification of human epidermal growth factor using surface plasmon resonance

Background: A method for the selection of suitable molecular recognition element (MRE) for the quantification of human epidermal growth factor (hEGF) using surface plasmon resonance (SPR) is presented. Two types of hEGF antibody, monoclonal and polyclonal, were immobilized on the surface of chip and validated for its characteristics and performance in the quantification of hEGF. Validation of this analytical procedure was to demonstrate the stability and suitability of antibody for the quantification of target protein. Results: Specificity, accuracy and precision for all samples were within acceptable limit for both antibodies. The affinity and kinetic constant of antibodies-hEGF binding were evaluated using a 1:1 Langmuir interaction model. The model fitted well to all binding responses simultaneously. Polyclonal antibody (pAb) has better affinity (KD = 7.39e-10 M) than monoclonal antibody (mAb) (KD = 9.54e-9 M). Further evaluation of kinetic constant demonstrated that pAb has faster reaction rate during sample injection, slower dissociation rate during buffer injection and higher level of saturation state than mAb. Besides, pAb has longer shelf life and greater number of cycle run. Conclusions: Thus, pAb was more suitable to be used as a stable MRE for further quantification works from the consideration of kinetic, binding rate and shelf life assessment

Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611

Background: Inulinase is a versatile enzyme from glycoside hydrolase family which targets the \u3b2-2, 1 linkage of fructopolymers. In the present study, the effect of medium composition and culture conditions on inulinase production by Aspergillus niger ATCC 20611 was investigated in shake-flasks. Results: The highest extracellular inulinase (3199 U/ ml) was obtained in the presence of 25% (w/v) sucrose, 0.5% (w/v) meat extract, 1.5% (w/v) NaNO3 and 2.5 mM (v/v) Zn2+, at initial pH of 6.5, temperature 35\ubaC and 6% (v/v) of spores suspension in the agitation speed of 100 rpm. Surfactants showed an inhibitory effect on enzyme production. The optimum temperature for inulinase activity was found to be 50\ubaC. TLC analysis showed the presence of both exo- and endo-inulinase. Conclusion: Sucrose, Zn2+, and aeration were found to be the most effective elements in inulinase production by A. niger ATCC 20611. TLC analysis also showed that the crude enzyme contained both endo and exoinulinases. The strain is suggested as a potential candidate for industrial enzymatic production of fructose from inulin

Aeration and yeast extract requirements for kojic acid production by Aspergillus flavus link

Growth and kojic acid production by Aspergillus flavus Link 44-1 were studied at different levels of dissolved oxygen tension (DOT) using a 2-l stirred tank fermenter. In all experiments, agitation was fixed at 600 rpm and DOT was controlled at different levels by varying airflow rates. Single-phase DOT control at three different levels (30, 50, and 80% saturation) did not enhance kojic acid production when compared to fermentation without DOT control (13.5 g l-l). The production of kojic acid in a fermentation with single-phase DOT control at 80% was comparable to that of a fermentation without DOT control. Decreased DOT levels below 80% reduced the production of kojic acid significantly although the biomass increased. When DOT was controlled at a very high level (80%) during active growth and then decreased to a low level (30%) during the production phase (i.e., two-phase DOT control), the production of kojic acid (28.9 g l-l) was increased by about two times compared to a fermentation without DOT control; however, when yeast extract was added continuously in a fermentation with two-phase DOT control, active growth occurred during the production phase and the maximum accumulation of kojic acid was reduced significantly. High DOT during on active growth phase and no addition of yeast extract during the production phase were required to enhance kojic acid biosynthesis. The most effective control strategy for kojic acid production, therefore, was to control DOT at very high levels during active growth and not add a nitrogen source during the production phase while using low levels of DOT

pH Shift Solubilization and Precipitation Protein Extraction from the Wastes of the Threadfin Bream, Nemipterus japonicus

This study aimed at extraction of protein from the wastes of the threadfin bream, Nemipterus japonicus using pH shift solubilization and precipitation processes and evaluation of the influencing factors on protein extraction. Towards this objective, the wastes of head, skin and internal organs were collected, and their nutritional composition evaluated. Furthermore, the ratio of waste: water for protein extraction was screened and the effect of pH, centrifugation speed and time on protein solubility rate, the amino acid composition and the molecular weight of proteins via SDS-PAGE were determined. Results demonstrated that moisture was the highest percentage (~70-78%) of all wastes. Highest protein content (19.67±1.10%), fat content (1.81±0.09%) and carbohydrate content (4.43±0.23%) were observed in skin, head and internal organs, respectively. The optimum ratio of waste: water for protein extraction is 1:9 for head, 1:8 for internal organs and 1:6 for skin. The protein extraction efficiency for all three samples were high at pH 3 and pH 12. Protein solubility increased with increased centrifugation speed up to 10,000 ×g, and there was no significant difference (P>0.05) between the protein solubility at 10,000 ×g and 20’000 ×g. Furthermore, there was significant difference (P<0.05) between protein solubility with increased centrifugation time. Alkaline process showed a remarkably higher amino acid content as compared to that of acid version. Glutamic acid and lysine were found higher compared to other amino acids. The molecular weight of proteins isolated in this study were low (<100 KDa)

A panel of cultivate specific marker based on polymorphisms at microsatellite markers for Iranian cultivated almonds (Prunus dulcis).

Abstract Molecular markers developed for Prunus also offer a powerful tool to study the evolution of the genome, and for understanding of genome structure and determinants of genetic diversity. Two hundred eighty almond genotypes/cultivars from different origins distributed throughout Iran besides some foreign cultivars and their hybrids with Iranian ones were collected. Microsatellite analysis was carried out using 9 pair flanking SSR sequences previously cloned and sequenced specifically in almond. The total number of detected alleles was 152 (9 to 20 alleles per locus with an average of 16.87). The mean PIC value of the polymorphic loci was relatively high (0.81) and the mean value for H e was 0.83, so that we were able to distinguish 98% of the genotypes using 5 loci. In cluster analysis, the genotypes were divided into 2 major groups, foreign cultivars and Iranian almond genotypes. Principal coordinate analysis based on Shared Allele method indicated proper distribution of the studied markers through the genome. Some specific markers were recorded among the germplasm which can be used efficiently in rapid and precise identification of the related genotypes and also in breeding programs through MAS. Genotypes were coded using our suggested coding method for genotype molecular identification

Extractive Fermentation for Recovery of Bacteriocin-Like Inhibitory Substances Derived from Lactococcus lactis Gh1 Using PEG2000/Dextran T500 Aqueous Two-Phase System

This work aimed to optimize the parameters affecting partitioning of a bacteriocin-like inhibitory substances (BLIS) from Lactococcus lactis Gh1 in extractive fermentation using polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS). This system was developed for the simultaneous cell cultivation and downstream processing of BLIS. Results showed that the molecular weight of PEG, PEG concentration, and dextran T500 affect the partition coefficient (K), purification factor (PF), and yield of BLIS partitioning. ATPS composed of 10% (w/w) PEG2000 and 8% (w/w) dextran T500, provided the greatest conditions for the extractive BLIS production. The K (1.00±0.16), PF (2.92±0.37) and yield (77.24±2.81%) were increased at selected orbital speed (200 rpm) and pH (pH 7). Sustainable growth of the cells in the bioreactor and repeated fermentation up to the eighth extractive batch were observed during the scale up process, ensuring a continuous production and purification of BLIS. Hence, the simplicity and effectiveness of ATPS in the purification of BLIS were proven in this study

Pasteurellosis vaccine commercialization: Physiochemical factors for optimum production

Pasteurella spp. are Gram-negative facultative bacteria that cause severe economic and animal losses. Pasteurella-based vaccines are the most promising solution for controlling Pasteurella spp. outbreaks. Remarkably, insufficient biomass cultivation (low cell viability and productivity) and lack of knowledge about the cultivation process have impacted the bulk production of animal vaccines. Bioprocess optimization in the shake flask and bioreactor is required to improve process efficiency while lowering production costs. However, its state of the art is limited in providing insights on its biomass upscaling, preventing a cost-effective vaccine with mass-produced bacteria from being developed. In general, in the optimum cultivation of Pasteurella spp., production factors such as pH (6.0–8.2), agitation speed (90–500 rpm), and temperature (35–40 °C) are used to improve production yield. Hence, this review discusses the production strategy of Pasteurella and Mannheimia species that can potentially be used in the vaccines for controlling pasteurellosis. The physicochemical factors related to operational parameter process conditions from a bioprocess engineering perspective that maximize yields with minimized production cost are also covered, with the expectation of facilitating the commercialization process