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FEMS Microbiology Letters, 362, 2015, fnv167
doi: 10.1093/femsle/fnv167
Advance Access Publication Date: 16 September 2015
Research Letter
R E S E A R C H L E T T E R – Biotechnology & Synthetic Biology
Characterizing and optimizing magnetosome
production of Magnetospirillum sp. XM-1 isolated
from Xi’an City Moat, China
Yinzhao Wang1,2 , Wei Lin1,2 , Jinhua Li1,2 , Tongwei Zhang1,2 , Ying Li3 ,
Jiesheng Tian3 , Lixin Gu4 , Yvan Vander Heyden5 and Yongxin Pan1,2,4,∗
1
Paleomagnetism and Geochronology Laboratory, Key Laboratory of Earth and Planetary Physics, Institute of
Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China, 2 France-China
Bio-Mineralization and Nano-Structures Laboratory, Institute of Geology and Geophysics, Chinese Academy of
Sciences, Beijing 100029, China, 3 State Key Laboratories for Agro-biotechnology and College of Biological
Sciences, China Agricultural University, Beijing 100094, China, 4 Electron Microscope Laboratory, Institute of
Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China and 5 Department of Analytical
Chemistry and Pharmaceutical Technology, Vrije Universiteit Brussel-VUB, Brussels 1050, Belgium
∗ Corresponding author: Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China. Tel: 86-010-82998406;
E-mail: [email protected]
One sentence summary: Characterization of a newly isolated magnetotactic bacteria and optimization of magnetosome production using statistic
experiment design and magnetic measurements.
Editor: Robert Gunsalus
ABSTRACT
Pure culture of magnetotactic bacteria is desirable to understand their physiology, evolution and biomineralization. Here,
we report a new strain Magnetospirillum sp. XM-1 that was recently isolated and cultivated from the eutrophic city moat of
Xi’an, China. Magnetosome biomineralization, crystallographic and magnetic properties of XM-1 were characterized by
using a combination of transmission electron microscopy and rock magnetic methods. Cell growth and magnetite
production was optimized by response surface methodology. We found that the Magnetospirillum strain XM-1 is different
from the model strain Magnetospirillum magneticum AMB-1 in terms of magnetite magnetosomes, optimal growth
temperature and nutrient requirements. Sodium succinate, sodium nitrate and ferric citrate are the three most significant
factors associated with the optimization of cell growth and magnetosome production for XM-1.
Keywords: Magnetospirillum sp. XM-1; magnetotactic bacteria; magnetic properties; magnetite formation; magnetosome
production; response surface methodology optimization
INTRODUCTION
Magnetotactic bacteria (MTB) strains of Magnetospirillum within
the Alphaproteobacteria are considered model organisms for magnetite magnetosome biomineralization. Among a group of Mag-
netospirillum strains, so far only four strains (M. magnetotacticum
strain MS-1, M. magneticum AMB-1, M. gryphiswaldense MSR-1
and Magnetospira sp. QH-2) have been well described (Blakemore,
Maratea and Wolfe 1979; Matsunaga, Tsujimura and Kamiya
1996; Schüler and Baeuerlein 1996; Zhu et al. 2010; Lefèvre
Received: 22 July 2015; Accepted: 13 September 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2015, Vol. 362, No. 21
et al. 2012; Bazylinski et al. 2013). Because the biotechnological
applications of magnetosome magnetite have been widely
considered in nanomaterial for magnetic separation, cancer detection, magnetic targeting hyperthermia, toxic metal bioremediation and even in fields such as magnetic sensors and magnetic memory (Matsunaga et al. 2007; Faivre and Schüler 2008;
Staniland et al. 2008; Alphandéry et al. 2011; Prozorov et al. 2013),
cultivation and characterization of new MTB strains from nature environments are desirable. Efforts on the cultivation of
MTB suggest that magnetosomes synthesized by different MTB
strains are diverse; thus, they may have specific utilization in
various domains (Yang et al. 2001; Heyen and Schüler 2003;
Zhang et al. 2011; Silva et al. 2013). In the present study, we combined response surface methodology (RSM), an efficient mathematical and statistical experimental strategy, to define the optimal medium composition and the best productive conditions
at certain prerequisite (Dejaegher and Vander Heyden 2011), and
of rock magnetic measurements (RMM) to cultivate and characterize the novel isolated Magnetospirillum sp. XM-1. The corresponding biomineralization process was further monitored by
using a combination of transmission electron microscopy (TEM)
and RMM methods.
Rock magnetic and ferromagnetic resonance
measurements
MATERIALS AND METHODS
Optimizing cell growth and magnetosome production
Isolation and cultivation of Magnetospirillum sp. XM-1
We used single factor experiments to select the best components and concentration ranges of carbon, nitrogen, sulfur and
iron sources within the culture medium after 96 hours incubation. Plackett–Burman experimental design and central composite design (CCD) were used for the RSM tests (Dejaegher and
Vander Heyden 2009). The responses, i.e. saturation magnetization (magnetite production), saturation magnetization per dry
weight (magnetite productivity) and coercivity, were analyzed
by analysis of variance (ANOVA). In order to generate a uniform combined model, we mathematically merged the three
responses into one response, named Desirability (Myers and
Montgomery 2002). Contour maps of the model for the combined
response were generated from Design-expert (version 8.0).
The surface sediment samples were taken from the city moat
of Xi’an, Shaanxi, China (34◦ 15 10.00 N, 108◦ 55 13.41 E). The detailed physical and chemical parameters of the water and sediment have been elaborated in Wang et al. (2013). Live MTB were
enriched by bar magnets and was transferred into several sterilized glass capillaries for magnetic purification (Wolfe, Thauer
and Pfennig 1987). Purified cell suspension was directly plated
onto a magnetic spirillum growth medium (MSGM) agar plate.
After 10 days of anaerobic incubation at 28◦ C in an anaerobic
chamber (ShangHai CIMO Medical Instrument Co. China), several round-shaped colonies with a dark brown color formed and
were suspended in liquid MSGM medium for further analyses
and stock.
Phylogenetic analysis of 16S rRNA gene sequence
One microliter of cultivated bacteria suspension was used for
16S rRNA gene amplification (Wang et al. 2013). Purified PCR
products ligated with the pMD19-T vector were transferred into
DH5α competent cells. After 15 hours incubation at 37◦ C, 40
colonies were selected randomly and sequenced. All sequences
were checked and analyzed by the Blastn program on the NCBI
website. Then representative sequences were aligned using
ClustalW program with other typical MTB 16S rRNA genes selected on the NCBI database (Thompson, Higgins and Gibson
1994). The phylogenetic tree was constructed using the MEGA
version 6.0 by neighbor-joining method with bootstrap repeated
1000 times (Tamura et al. 2013).
TEM analysis
Approximately 20 μL of cell suspension and 5 μL of isolated
magnetosome suspension were transferred onto carbon-coated
copper grids, respectively. Cell morphology, magnetosome structure, energy dispersive X-ray spectroscopy (EDXS), fast Fourier
transform (FFT) pattern and selected area electron diffraction
(SAED) pattern analyses were performed by a JEM-2010 electron
microscope (200 kV) (JEOL, Japan).
Fresh whole cells were collected by centrifugation at 8000 rpm
10 min at 4◦ C, and the cell pellets were immediately transferred into a COY anaerobic chamber ([O2 ] < 300 ppm, COY7000220A, Coy Laboratory Products, USA), loaded into nonmagnetic gelatin capsules and dried for 24 h. Hysteresis loops,
first-order reversal curves (FORCs) and saturation isothermal
remanent magnetization (SIRM) were carried out on a vibrating sample magnetometer Model 3900 (Princeton Measurements
Corporation, USA, sensitivity 5.0 × 10−10 Am2 ) and saturation
magnetization (Ms ), saturation remanence (Mrs ), coercivity (Bc )
and remanence coercivity (Bcr ) were determined. Zero field cooling (ZFC) and field cooling (FC) experiments were performed
on a Quantum Design Magnetic Property Measurement System
(MPMS XP-5). The Verwey transition temperature (Tv ), δ FC , δ ZFC
and δ-ratio (δ FC /δ ZFC ) were calculated according to Moskowitz,
Frankel and Bazylinski (1993). Ferromagnetic resonance (FMR)
measurements were performed with a JEOL ESR FA-200 spectrometer at a microwave frequency of 9.0 GHz at room temperature.
Batch cell culture and pH-static fermentation
Batch cell cultures were grown within 4 L optimized culture
medium in 6-L screw-sealed glass bottles with optimized parameters. For pH-static culture, the cells were grown in a 7-L fermenter (BioTECH, Shanghai, China) with 4.5 L liquid medium.
Before fermentation, effects of different yeast extract contents
in optimized medium were tested. Then, optimized medium
(with yeast extract) was used for evaluation of cell growth
and magnetosome production. Fermentation was maintained at
30◦ C and the pH was kept constant at 7.2 by automated supplementation of 0.5 M HCl. Filter-sterilized air and nitrogen gas
with per-mixed ratio of 1:10 and an initial airflow of about 0.05
L min−1 was used in the culture system and the dissolved O2
was maintained below 1%. Agitation was set at 100 rpm after 12
h static growth. During the cultivation, 50 ml of cell suspension
was sampled for TEM, cell growth and RMM tests at different
time interval.
Cell density and nutrients consumption
The cell growth curve was monitored by absorbance measurements at 600 nm (OD600 ). The pH value of the culture medium
was measured by a Mettler Toledo Delta 320 meter (MettlerToledo Greifensee, Switzerland). The concentrations of nutrient
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Figure 1. A neighbor-joining tree with 1000 bootstrap values displays the phylogenetic relationship between strain XM-1 and other typical MTB based on 16S rRNA
gene sequences.
components in the culture mediums were measured after removing all the cells by centrifugation and filtrating with a 0.22μm filter. Specifically, the concentration of succinate within the
medium was measured according to the succinate kit procedure (Megazyme, Ireland) and detected by a microplate reader
(SpectraMax i3, Molecular Devices Corporation, USA). Nitrate
and total iron were measured using a DR2800 Spectrophotometer (HACH, Loveland, Colorado, USA) and powder pillows detection kits (HACH, Loveland, Colorado, USA) based on the cadmium reduction method and FerroMo method, respectively, according to the manufacturer’s instructions.
RESULTS
Isolation, cultivation and phylogenetic lineage
Dark, single colonies with round shape formed after 10 days
growth of magnetically separated MTB cells on solid agar plates
under anaerobic condition at 28◦ C. Optical and electron microscope checks showed that these isolated cells were 1–5 μm
vibrioid-to-helical shaped (Fig. 2A and G). Phylogenetic analysis
of 16S rRNA genes showed the newly isolated MTB, tentatively
named XM-1, belonged to the family Rhodospirillaceae of the class
Alphaproteobacteria (Fig. 1).
Characterization of magnetosome produced by strain
XM-1
TEM observations show that XM-1 synthesizes a single intact
chain with 10 magnetosomes per cell on average with mean
size and shape factor of 43.7 nm and 0.85, respectively (Fig. 2H
and I). High-resolution TEM (HRTEM) observations reveal that
magnetosomes are truncated, octahedral magnetite (i.e. slightly
elongated cuboctahedrons) (Fig. 2B–E). The EDXS, SEAD pattern
and lattice space data from the isolated magnetosomes indicate magnetite (Fe3 O4 ) composition of the XM-1 magnetosome
(Fig. 2F and Fig. S1, Supporting Information).
The FMR spectrum of XM-1 cells has two shoulders at 160 and
291 mT (Fig. 3A). The spectral parameter Beff and geff are 360.53
mT and 1.78, respectively. The line width BFWHM is measured
as 190.56 mT, and B are 34.5 and 156.8 mT for the low-field
and high-field absorption, respectively. The asymmetry ratio A
is 0.221.
Hysteresis loop of anaerobically dried XM-1 cells shows a typical Stoner-Wohlfarth type loop with Bc , Bcr , Bcr /Bc and Mrs /Ms
of 30.98 mT, 41.78 mT, 1.35 and 0.47, respectively (Fig. 3C). The
FORC diagram (Fig. 3E) is nicely characterized by a rather narrow
distribution around Hc,FORC ∼38 mT along the horizontal axis,
so-called central ridge distribution for non-interacting magnetosome chains (Egli et al. 2010). Both FC and ZFC thermal demagnetization curves of SIRM10K˙2.5T show drops in remanence
between 90 and 112 K, and the calculated Tv , δ FC , δ ZFC and δ-ratio
were 98 K, 0.147, 0.039 and 3.8, respectively (Fig. 3B). The cross
of IRM acquisition and demagnetization curves is 0.47 (Fig. 3D),
showing a very weak magnetic interaction.
RSM medium optimization
A total of 11 factors were determined by single factor experiments by cell growth and magnetosome content. Their influences on magnetite production, productivity and coercivity were
scanned using a Plackett–Burman design (Tables S1 and S2,
Supporting Information). High coercivity values usually indicate
larger grain sizes of magnetite or longer chain configuration or
both in whole cells. NaNO3 , sodium succinate and ferric citrate
are determined to be the most significant factors on the three
analyzed responses, whereas, for other components, the final
concentrations are defined by their specific positive or negative
contribution to each response even though they are not significant (see factors in Table S1, Supporting Information, with asterisks).
Results of the CCD experiment factors and ANOVA results
calculated from the combined response are shown in Fig. 4 and
Tables S3–S5 (Supporting Information). The optimal concentrations for the three factors derived from the model are 17.32 mM
for NaNO3 , 20.18 mM for sodium succinate and 2.13 mM for ferric citrate, which is template named as XM-C medium. With the
XM-C medium, we got magnetite production, magnetite productivity and coercivity of 0.13 mAm2 /L, 0.65 mAm2 /g and 30.01 mT,
respectively. The result agreed well with the model prediction
values (see Table S6, Supporting Information). We also compared
the results using MSGM and enhanced MSGM culture medium
with the optimal growth medium in 1 L sealed glass bottles. The
results showed that the optimal process conditions significantly
increased nearly 5-fold regarding the cell yield and magnetite
production of strain XM-1.
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FEMS Microbiology Letters, 2015, Vol. 362, No. 21
Figure 2. TEM analyses of the XM-1. (A) Typical TEM micrograph of strain XM-1; (B) magnetosome imaged by HRTEM (crystal structure is shown by white line along the
magnetosome); (C) calculated 3D model viewed from 011 axis; (D) FFT pattern of the crystal; (E) and (F) the TEM picture and SEAD pattern of the extracted magnetosome,
respectively. Plots (G), (H) and (I) show the histograms of cell length, magnetosome number per cell and magnetosome size of strain XM-1, respectively.
Growth, magnetosome production and nutrients
consumption
As shown in Fig. 5A, magnetite production (saturation magnetization per liter) increased dramatically at exponent phase of
cell growth, and showed a similar pattern as the cell growth
curve, indicating that the magnetite crystallization rate during the exponent phase was correlated with the cell reproduction rate in optimized batch culture conditions. Notably, the pH
of the culture medium increased during growth, corresponding to the consumption of succinate, nitrate and total iron
(Fig. 5B). In order to further examine the magnetosome production, the growth in a pH and oxygen stat 7 L fermenter was carried out. Single factor experiment was preformed on different
concentrations of yeast extracts added into the optimized culture, and 1.6 g/L yeast extracts showed the highest cell and magnetosome yields then defined as XM-C-YEx. Surprisingly, in a pH
static fermenter, the cell growth increased to a maximum point
(0.37 g/L cell dry weight, 7.1 mg/L magnetosome dry weight)
within 48 h growth (Fig. 5C).
DISCUSSION
Specific environments are usually dominated by MTB of related
metabolism preference (Lins et al. 2007; Lin and Pan 2010; Lefèvre
et al. 2012; Lin et al. 2013; Chen et al. 2015). Despite the studied strain XM-1 showed a 99% 16S rRNA gene similarity with
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Figure 3. Magnetic properties of XM-1 cell samples. Ferromagnetic resonance (A), low temperature magnetic measurements (B), room-temperature hysteresis loop (C),
Wohlfarth–Cisowski test (D) and FORC diagram (E).
strain AMB-1, the newly isolated strain synthesizes ∼10 magnetite magnetosomes arranged in a single magnetosome chain
and has an optimal growth temperature 4◦ C higher than AMB-1 s
(Yang et al. 2001). Strain XM-1 also prefers higher iron and nutrients concentration, which benefits both cell yield and magnetosome production compared with AMB-1 (Matsunaga, Tsujimura
and Kamiya 1996; Yang et al. 2001). It is assumed that the eutrophic city moat may endow strain XM-1 the ability to tolerate
and adapt to high nutrients concentration compared with other
magnetotactic spirilla isolated from oligotrophic water areas.
However, strain XM-1 only showed slight differences in magnetic properties compared with other studied strains, e.g. MS-1,
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Figure 5. Time courses of XM-1 cell growth, medium chemical change and magnetosome formation. (A) Cell growth (OD600 , displayed as dots), general magnetosome production (saturation magnetization, displayed as gray bars) and coercivity (displayed as triangles) during the growth of strain XM-1 in the optimized culture medium. (B) pH changes (dots), sodium succinate (squares), NaNO3 (triangles) and ferric citrate (crosses) concentration during cell growth. (C) Cell growth
(OD600 , displayed as dots), general magnetosome production (saturation magnetization, displayed as gray bars) and coercivity (displayed as triangles) during the
growth in a 7 L fermenter with XM-C-YEx growth media.
Figure 4. Contour diagrams from the model that generated with the results of
the CCD set-up. For the combined response, named desirability was obtained
by merging magnetosome production, magnetosome productivity and coercivity
results mathematically. (A–C) show the contour plots for the concentrations of
sodium succinate and NaNO3 (ferric citrate at 2.13 mM), ferric citrate and NaNO3
(sodium succinate concentration at 20.18 mM), and ferric citrate and sodium
succinate (NaNO3 at 17.32 mM), respectively.
MSR-1 and QH-2, within the same genus, indicating that in general the synthesis of magnetite magnetosomes within these
magnetotactic spirilla is mostly biologically controlled through
clusters of magnetosome genes (Kobayashi et al. 2006; Faivre and
Schüler 2008; Carvallo et al. 2009; Zhu et al. 2010; Komeili 2012; Li
and Pan 2012; Siponen et al. 2013).
For evaluation of magnetite magnetosome production, we
used the magnetic parameter of saturation magnetization per
liter of whole cells. We measured the whole cells and magnetosome dry weights and their saturation magnetization (Ms ). Both
cell and magnetosome dry weight exhibit a very good linearity (Fig. 6, R2 magnesome = 0.9902; R2 cell dry weight = 0.9946) with the
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ACKNOWLEDGEMENTS
The authors thank two anonymous reviewers for their constructive comments, Artemis Louyakis at Space Life Sciences Lab,
University of Florida for improving the English usage and Yang
Sun at Institute of Physics, CAS for kind help in ferromagnetic
resonance analysis.
FUNDING
This study was supported by the Chinese Academy of Sciences
project and National Natural Science Foundation of China grant
[grant number 41330104].
Conflict of interest. None declared.
REFERENCES
Figure 6. The relationships between saturation magnetization and (A) magnetosome dry weight, and (B) cell dry weight. The saturation magnetization shows
very good linearity with both magnetosome and cell dry weights.
Ms , suggesting Ms as a good proxy for cell growth and magnetosome production. Thus, we can estimate the magnetosome
mass from magnetic measurement with only a small amount
of cells through the theoretical magnetite saturation magnetization (92 mAm2 /g) (O’Reilly 1984). Based on Ms , we estimated
the final magnetosome production at optimal culture as 1.44 mg,
which is close to the actual weight of 1.39 mg. In contrast to
Ms , Bc , which is independent of concentration, is mainly related
to the magnetic domain state (size) and particle interaction of
magnetosomes (Pan et al. 2005; Carvallo et al. 2009; Li et al. 2009).
Therefore, we propose that rock magnetic parameters are useful
proxies to evaluate magnetite magnetosome both qualitatively
and quantitatively, and thus could be considered as a uniform
standard approach for magnetosome production assessment.
Although magnetite magnetosome production in fermentation system increased nearly 20-fold compared with cultivation
using MSGM and enhanced MSGM culture media, the cell and
magnetosome dry weight per liter was still not very high in
comparison with that of strain MSR-1 and MV-1 (Zhang et al.
2011; Silva et al. 2013). One possible explanation is that the combined response is not representing the desirability of the analyst. For example, the optimization results in a large increase in
magnetite magnetosome production, while only a limited influence on the responses coercivity is seen (Dejaegher and Vander Heyden 2009). Alternatively, the newly isolated MTB may
not have completely acclimated to laboratory conditions. In future research, precise oxygen flow control and nutrient feedback
should be considered to improve the magnetosome productive
ability in a fermentation system as well.
Nucleotide sequence accession numbers
The sequence data were submitted to the DDBJ/EMBL/GenBank
databases under accession numbers KP966105.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSLE online.
Alphandery E, Faure S, Seksek O, et al. Chains of magnetosomes
extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy. ACS Nano
2011;5:6279–96.
Bazylinski DA, Williams TJ, Lefèvre CT, et al. Magnetococcus marinus gen. nov., sp nov., a marine, magnetotactic bacterium
that represents a novel lineage (Magnetococcaceae fam. nov.,
Magnetococcales ord. nov.) at the base of the Alphaproteobacteria. Int J Syst Evol Micr 2013;63:801–8.
Blakemore RP, Maratea D, Wolfe RS. Isolation and pure culture
of a freshwater magnetic spirillum in chemically defined
medium. J Bacteriol 1979;140:720–9.
Carvallo C, Hickey S, Faivre D, et al. Formation of magnetite
in Magnetospirillum gryphiswaldense studied with FORC diagrams. Earth Planets Space 2009;61:143–50.
Chen Y, Zhang R, Du H, et al. A novel species of ellipsoidal
multicellular magnetotactic prokaryotes from Lake Yuehu in
China. Environ Microbiol 2015;17:637–47.
Dejaegher B, Vander Heyden Y. The use of experimental design
in separation science. Acta Chromatogr 2009;21:161–201.
Dejaegher B, Vander Heyden Y. Experimental designs and their
recent advances in set-up, data interpretation, and analytical
applications. J Pharmaceut Biomed 2011;56:141–58.
Egli R, Chen AP, Winklhofer M, et al. Detection of noninteracting single domain particles using first-order reversal curve
diagrams. Geochem Geophy Geosy 2010;11:Q01Z11.
Faivre D, Schüler D. Magnetotactic bacteria and magnetosomes.
Chem Rev 2008;108:4875–98.
Heyen U, Schüler D. Growth and magnetosome formation
by microaerophilic Magnetospirillum strains in an oxygencontrolled fermentor. Appl Microbiol Biot 2003;61:536–44.
Kobayashi A, Kirschvink JL, Nash CZ, et al. Experimental observation of magnetosome chain collapse in magnetotactic bacteria: Sedimentological, paleomagnetic, and evolutionary implications. Earth Planet Sci Lett 2006;245:538–50.
Komeili A. Molecular mechanisms of compartmentalization and
biomineralization in magnetotactic bacteria. FEMS Microbiol
Rev 2012;36:232–55.
Lefèvre CT, Schmidt ML, Viloria N, et al. Insight into the evolution
of magnetotaxis in Magnetospirillum spp., based on mam gene
phylogeny. Appl Environ Microb 2012;78:7238–48.
Li J, Pan Y. Environmental factors affect magnetite magnetosome synthesis in Magnetospirillum magneticum AMB-1: Implications for biologically controlled mineralization. Geomicrobiol J 2012;29:362–73.
8
FEMS Microbiology Letters, 2015, Vol. 362, No. 21
Li J, Pan Y, Chen G, et al. Magnetite magnetosome and fragmental chain formation of Magnetospirillum magneticum AMB1: transmission electron microscopy and magnetic observations. Geophys J Int 2009;177:33–42.
Lin W, Pan Y. Temporal variation of magnetotactic bacterial
communities in two freshwater sediment microcosms. FEMS
Microbiol Lett 2010;302:85–92.
Lin W, Wang Y, Gorby Y, et al. Integrating niche-based process
and spatial process in biogeography of magnetotactic bacteria. Sci Rep 2013;3:1643.
Lins U, Keim CN, Evans FF, et al. Magnetite (Fe3 O4 ) and greigite
(Fe3 S4 ) crystals in multicellular magnetotactic prokaryotes.
Geomicrobiol J 2007;24:43–50.
Matsunaga T, Suzuki T, Tanaka M, et al. Molecular analysis
of magnetotactic bacteria and development of functional
bacterial magnetic particles for nano-biotechnology. Trends
Biotechnol 2007;25:182–8.
Matsunaga T, Tsujimura N, Kamiya S. Enhancement of magnetic particle production by nitrate and succinate fedbatch culture of Magnetospirillum sp. AMB-1. Biotechnol Tech
1996;10:495–500.
Moskowitz BM, Frankel RB, Bazylinski DA. Rock magnetic criteria
for the detection of biogenic magnetite. Earth Planet Sci Lett
1993;120:283–300.
Myers RH, Montgomery DC. Response Surface Methodology. New
York: Wiley, 2002.
O’Reilly W. Rock and Mineral Magnetism. Glasgow: Blackie, 1984.
Pan Y, Petersen N, Winklhofer M, et al. Rock magnetic properties of uncultured magnetotactic bacteria. Earth Planet Sci Lett
2005;237:311–25.
Prozorov T, Bazylinski DA, Mallapragada SK, et al. Novel magnetic nanomaterials inspired by magnetotactic bacteria: Topical review. Mat Sci Eng R 2013;74:133–72.
Schüler D, Baeuerlein E. Iron-limited growth and kinetics of
iron uptake in Magnetospirillum gryphiswaldense. Arch Microbiol 1996;166:301–7.
Silva KT, Leão PE, Abreu F, et al. Optimized magnetosome production and growth by the magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 using statistical experimental design. Appl Environ Microb 2013;79:2823–37.
Siponen IM, Legrand P, Widdrat M, et al. Structural insight into
magnetochrome-mediated magnetite biomineralization. Nature 2013;502:681–4.
Staniland S, Williams WYN, Telling N, et al. Controlled cobalt
doping of magnetosomes in vivo. Nat Nanotechnol 2008;3:
158–62.
Tamura K, Stecher G, Peterson D, et al. MEGA6: Molecular
Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol
2013;30:2725–9.
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:
4673–80.
Wang Y, Lin W, Li J, et al. High diversity of magnetotactic
Deltaproteobacteria in a freshwater niche. Appl Environ Microb
2013;79:2813–7.
Wolfe RS, Thauer RK, Pfennig N. A capillary racetrack method
for isolation of magnetotactic bacteria. FEMS Microbiol Ecol
1987;45:31–5.
Yang CD, Takeyama H, Tanaka T, et al. Effects of growth medium
composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum
AMB-1. Enzyme Microb Tech 2001;29:13–9.
Zhang Y, Zhang X, Jiang W, et al. Semicontinuous culture of Magnetospirillum gryphiswaldense MSR-1 cells in an autofermentor
by nutrient-balanced and isosmotic feeding strategies. Appl
Environ Microb 2011;77:5851–6.
Zhu K, Pan H, Li J, et al. Isolation and characterization of a marine
magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea. Res Microbiol 2010;161:276–83.