Semicontinuous Culture of Magnetospirillum gryphiswaldense MSR-1 Cells in an Autofermentor by Nutrient-Balanced and Isosmotic Feeding Strategies

Abstract

An improved strategy was developed for the high-density culture of Magnetospirillum gryphiswaldense strain MSR-1 and large-scale magnetosome production in both 7.5-and 42-liter autofermentors. By using a nutrientbalanced feeding strategy and the replacement of carbon and nitrogen sources to reduce accumulation of Na ؉ and Cl ؊ ions, we reduced the factors that tend to inhibit cell growth, particularly the increase of osmotic potential. Semicontinuous culture was thereby achieved in the autofermentor for the first time. When the cells were harvested at 36 and 73 h, magnetosome yields (dry weight) as high as 168.3 and 83.5 mg/liter/day, respectively, were achieved. These values were, respectively, approximately 10 and 5 times higher than the yields achieved in previous studies and represent a significant improvement in magnetosome production efficiency.Biomineralized magnetosomes (chains of magnetite crystals found in prokaryotes) have attracted commercial interest because of their narrow size range, good dispersibility, and biomembrane enclosure. Previous studies have addressed a variety of applications and properties, including enzyme immobilization (5), gene delivery system (16), cell separation (19), drug carriers (11, 12), immunoassays (4, 14), protein and multisubunit enzyme complexes (7,20), and use of microorganisms per se for mineral recovery (13). Because of the highly restrictive culture conditions for magnetotactic bacteria, in terms of the dissolved oxygen concentration (dO 2 ) (3, 17), nutrients, etc., the yields of both magnetosomes and their host microorganisms under artificial culture tend to be low (10, 18). A long-standing research goal of our laboratory is improved large-scale production of cells and magnetosomes.In a previous study using fed-batch culture techniques (10), we achieved maximal cell density (optical density at 565 nm [OD 565 ] of 7.24, cell dry weight of 2.17 g/liter [0.87 g/liter/day], and magnetosome dry weight of 41.7 mg/liter [16.7 mg/liter/ day]). Through further optimization of culture temperature, pH, dO 2 , and nutrients, we achieved an OD 565 value of 12 in a 7.5-liter fermentor after 40 h of culture (unpublished data). In revising our previous feeding strategy, we focused on supplementation of carbon and nitrogen sources but ignored two possible factors that could inhibit cell growth: (i) nutrient limitation arising during fermentation process and (ii) the accumulation of Na ϩ and Cl Ϫ in a fermentor fed with sodium lactate and ammonium chloride. By replacing the carbon and nitrogen sources and using an optimally nutrient-balanced feeding strategy, in a 7.5-liter fermentor after 44 h, we achieved an OD 565 of 30.4, a cell dry weight of 7.59 g/liter (3.8 g/liter/ day), and a magnetosome dry weight of 225.53 mg/liter (112.77 mg/liter/day). In a larger (42-liter) fermentor, after 44 h, we achieved an OD 565 of 42, a cell dry weight of 9.16 g/liter (4.58 g/liter/day), and a magnetosome dry weight of 356.52 mg/liter (178.26 mg/liter/day). The efficiency of magnetosome produ...

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium

“…Despite some progress by growth optimization (22,34), previous attempts to increase magnetosome yields at a large scale were met by only limited success. In this work, we established a strategy to enhance magnetosome production by genetic engineering.…”

Generation of Multifunctional Magnetic Nanoparticles with Amplified Catalytic Activities by Genetic Expression of Enzyme Arrays on Bacterial Magnetosomes

“…For instance, Zhang et al [47] reported high-density cultivation of M. gryphiswaldense in a large-scale fermentor. Furthermore, the overexpression of magnetosome biosynthesis genes by genomic multiplication in M. gryphiswaldense led to nearly threefold increased overproduction of magnetosomes.…”

Optimization of Magnetosome Production and Growth by the Magnetotactic Vibrio Magnetovibrio blakemorei Strain MV-1 through a Statistics-Based Experimental Design

“…Therefore, it seems likely that any of these growth factors, if necessary, are synthesized by M. blakemorei. Vitamin addition was also deemed unnecessary for the growth of M. gryphiswaldense, and thus, vitamins were eliminated from the growth medium in studies involving other magnetotactic bacteria (12). Our data indicated that high iron concentrations might have a potentially deleterious effect on M. blakemorei cell growth, but we retained it in the growth medium at a constant concentration (through injection of pulses of iron) since it is absolutely required for magnetosome biomineralization.…”