M. gryphiswaldense strain MSR-1 (DSM6361) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Brunswick, Germany). Seed culture was performed as described previously (Liu et al., 2010). The 42-L fermentor was filled with 30 L of medium containing 1.5 g sodium thioglycolate, 6 g magnesium sulfate heptahydrate, 15 g yeast extract, 6 g peptone, and 15 mL Wolfe's mineral solution. Feed medium contained 4.2 g ferric citrate, 129 g sodium lactate, 52.6 g LA, and 54.9 g ammonium chloride in a volume of 700 mL. MSR-1 cells were continuously cultured in the fermentor for 48 h, at which point the OD₅₆₅ value began to decrease following a long-term rise. The temperature and pH were controlled to 30°C and 6.8, respectively, during culture. The pH was adjusted via the feed medium. The dO₂ concentration was recorded using two probes that had different purposes. One probe was used for relative measurement (percent % as unit of data) and had lower accuracy. The other probe was used for absolute measurement (ppb as unit of data) and had higher accuracy. Calibration prior to measurements was required for the former probe but not for the latter. Before inoculation, we maintained an initial stirring rate (120 rpm) and airflow rate (0.94 L/min) for 2 h to ensure that oxygen was at saturation level in the medium. We then calibrated the dO₂ as closely as possible to 100% (there were occasional minor fluctuations). The various study parameters were recorded after inoculation as functions of time (Table 1).

Samples were taken every few hours for further analysis. After sampling, cell density (OD₅₆₅) and magnetism (C_mag) were measured immediately using the same spectrophotometer at wavelength 565 nm as described by Schüler et al. (1995). A 2-mL culture sample was centrifuged, and the LA concentration of the supernatant was measured immediately using a SBA-40C Biosensor analyzer (Institute of Biology, Shandong Academy of Sciences, China). Cells (in a defined volume) were washed twice with 50 mM Tris buffer (pH 7.0) and centrifuged at 4°C, and the cell pellets were stored at −80°C. Culture samples were suspended in an appropriate volume of sterile distilled water for subsequent measurements.

Another 2-mL culture sample was centrifuged in a 1.5-mL tube, and the pellet was dried at 60°C until it reached a constant weight. Iron content was measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES; model Optima 5300DV, Perkin Elmer, Waltham MA, USA). The percentage of iron in cells was calculated as the iron content divided by the dry weight.

Another 2-mL culture sample was added with 10% trichloroacetic acid solution (1:1, v/v), incubated on ice for 10 min, and centrifuged for 5 min at 4°C. The ATP content of the supernatant was measured using an AF-100 ATP analyzer (DKK-TOA Corp., Tokyo, Japan). The ATP content of the sample was calculated as total ATP content / volume of sample/OD₅₆₅ of sample.

Reducing power was measured by a modification of the method of Perez et al. (2008). For NADH measurement, a 1-mL sample was incubated with 50 μL KOH (0.4 mol/L, pH 12.3) for 10 min at 30°C, centrifuged (12,000 rpm) for 10 min at 4°C, and the supernatant was assayed. For NAD⁺ measurement, a 1-mL sample was incubated with 125 μL HCl (0.4 mol/L, pH 1.3) for 10 min at 5°C, centrifuged (12,000 rpm) for 10 min at 4°C, and the supernatant was assayed. The reducing power was calculated as the ratio of NADH to NAD⁺.

A culture sample was placed on a copper grid, washed twice with distilled water, dried, and observed with a transmission electron microscope (model JEM1230; JEOL, Tokyo, Japan).

Cells were lysed by ultrasonication (ice bath, 50 W, 200 times, 50% duty cycle). Cell debris was removed by centrifugation at 4°C to yield a supernatant (used as crude enzyme extract), and the protein content (mg) was measured by Coomassie Brilliant Blue assay (Chial et al., 1993). The total SOD activity was measured using a SOD assay kit (Jiancheng Corp., Nanjing, China). The SOD activity of a sample was calculated as the total SOD activity divided by protein content.

We performed several preliminary experiments to obtain stable results based on our past experience. Although there were slight differences in the data from different batches, the overall trends for cell growth rate and magnetosome yield were consistent (see Figures 1A, S1). The results from a representative experiment are shown in Figure 1 as an example. As showed in Figure 1, cells grew slowly (OD₅₆₅ ≤ 2.0) until hour 20. Thereafter, cell growth entered the log phase. Maximal values of OD₅₆₅ (19.4) (Figure 1A) and cell yield (dry weight) (4.76 g/L) were observed at hour 40. In MSR-1 culture, high dO₂ levels (>1%) promote cell growth but inhibit magnetosome formation (Heyen and Schüler, 2003; Liu et al., 2010). To balance these conflicting effects, we set the initial (hour 0) stirring rate to 120 rpm, the airflow rate to 0.94 L/min and the saturated dO₂ under these conditions to 100% (showed 102.7% after setting), based on the results of our previous study (Liu et al., 2010). After inoculation, the dO₂ level decreased rapidly to 0.0% at 8 h (Table 1). During this period, cell density (OD₅₆₅) increased 3-fold (from 0.1 at 0 h to 0.3 at 8 h), indicating a high oxygen requirement during the early stage of cell growth. To maintain a suitable dO₂ (0.0%) for magnetosome formation, we subsequently altered the stirring rate at 2-h intervals between 10 and 30 h and altered the airflow rate at 10, 12, 20, 22, and 32 h (Table 1). To precisely control dO₂ concentration, we used two probes with differing accuracies as described in Materials and Methods, one for relative dO₂ measurement (percent %), the other for absolute measurement (ppb). This approach allowed us to accurately detect even tiny changes of dO₂, particularly at extremely low concentrations. From 28 to 34 h, the absolute dO₂ level gradually increased nearly 23-fold, from 9 to 204 ppb, while the probe used for relative measurement continuously showed a value below the limit of detection (0.0%), which may have caused a delay in adjusting the stirring rate and airflow rate in the fermentor (Table 1). A high dO₂ concentration affected magnetosome formation. From 8 to 24 h, the absolute dO₂ concentration remained <20 ppb, while the C_mag value increased from 0.24 at 8 h to 1.78 at 20 h and remained high until 24 h (Figure 1B). dO₂ increased gradually from 17 ppb at 24 h to 1247 ppb at 40 h, while C_mag decreased from 1.71 at 24 h to 1.12 at 40 h. Taken together, these findings indicate that the optimal concentration of absolute dO₂ for magnetosome production is <20 ppb.

The LA feed medium contained LA, sodium lactate, ferric citrate, and ammonium chloride (see Materials and Methods). The LA in this medium was utilized both as a carbon source and a pH regulator. The supply of feed medium was auto-controlled via a computer to maintain a pH value of 6.8 throughout cell culture. The LA concentration in medium at each time point was recorded using a Biosensor analyzer and used as an indicator of the cell growth state. The LA concentration decreased rapidly from 0.26 g/L at 12 h to 0.01 g/L at 26 h, corresponding to the log phase of growth curve and reflecting the high cellular demand for LA as a source of carbon and energy for growth (Figure 1C). The LA concentration then increased from 0.01 g/L at 26 h to 0.16 g/L at 40 h. Excessive LA was present during this period and was associated with frequent pH fluctuations and rapid cellular metabolism (Figure 1C).

C_mag can be a useful parameter for estimating the magnetosome content of cells (Schüler et al., 1995), but it is based on an indirect method. A more direct method of measurement is based on TEM and the iron content of cells. The iron content in MTB can be 100-fold (or more) higher than in E. coli (Blakemore et al., 1979) and is concentrated primarily in magnetosomes (Kasama et al., 2006). We measured iron content by ICP-OES as described in Materials and Methods. C_mag and iron content both increased gradually from 8 to 20 h, reached maximal values (1.78 and 0.47%) at 20 h, and then decreased (to 1.12 and 0.20%, respectively) at 40 h (Figure 1D). TEM showed that the average number of magnetosomes per cell increased from 12 to 24 during the initial phase (0–20 h) and that magnetosome chains were not integrated at 12 h (Figures 2, S2 and Table 2). From 20 to 40 h, the average magnetosome number remained essentially constant (26 ± 3), and the chains were gradually integrated and matured (Figures 2, S2). We concluded that 20 h is an important transitional time point for magnetosome production. In view of the rapid decrease of LA content in medium around this time (Figure 1C), it is clearly important to provide sufficient nutritional supplements prior to 20 h to ensure the rapid growth of new cells.

The formation of magnetosomes in MTB is presumed to consume a large amount of energy. The availability of ATP as an energy source has been shown to be required for iron uptake in MTB (Nakamura et al., 1995; Schüler, 1999). We used an ATP analyzer to measure the ATP content of cells. From 0 to 20 h, the period of magnetosome synthesis, energy (i.e., ATP content) in cells decreased rapidly from 2.73 to 0.17 fmol. From 20 to 40 h, the ATP content continued to decrease from 0.17 fmol to near zero (0.02 fmol); however, the rate of decrease was lower than that from 0 h to 20 h (Figure 1E).

NADH plays an important role in ATP generation in cells and provides a proton gradient across the inner mitochondrial membrane for ATP production by ATP synthetase (Bonora et al., 2012). We used a modified enzyme reduction method to measure reducing power (as the ratio NADH/NAD⁺) (Perez et al., 2008). In contrast to the trend for ATP, the NADH/NAD⁺ ratio remained low (≤ 2) and fluctuated slightly during the first 20 h (Figure 1F). Thereafter, as the cells entered the log phase of growth and the magnetosomes matured, the NADH/NAD⁺ ratio increased rapidly to 15 at 40 h. These findings indicate that the process of magnetosome synthesis consumes a large amount of ATP and accumulates reducing power.

SOD in microbes helps remove the superoxide anion radical (O₂⁻) and hydrogen peroxide (H₂O₂), which have destructive effects on cell macromolecules (Cabiscol et al., 2000). In MTB, SOD also reduces oxidative stress during magnetosome formation. We used a SOD assay kit to monitor SOD activity in cells. The activity declined gradually from 555 U/mg at 8 h to 410 U/mg at 20 h and then rapidly to 164 U/mg at 40 h (Figure 1G).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.