Respiring cellular nano-magnets

Accepted Manuscript Respiring cellular nano-magnets

Ayesha Talib, Zanib Khan, Habib Bokhari, Syed Hidayathula, Ghulam Jilani, Abid Ali Khan PII: DOI: Reference:

S0928-4931(16)31040-2 doi: 10.1016/j.msec.2017.07.001 MSC 8150

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

29 August 2016 30 May 2017 3 July 2017

Please cite this article as: Ayesha Talib, Zanib Khan, Habib Bokhari, Syed Hidayathula, Ghulam Jilani, Abid Ali Khan , Respiring cellular nano-magnets, Materials Science & Engineering C (2017), doi: 10.1016/j.msec.2017.07.001

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ACCEPTED MANUSCRIPT Respiring Cellular Nano-Magnets Ayesha Talib1, Zanib Khan2, Habib Bokhari1, Syed Hidayathula3, Ghulam Jilani4 and Abid Ali Khan*1 1

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Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Tarlai Kalan, 45550 Islamabad, Pakistan 2 Department of Microbiology, Government Post Graduate College No. 2, Mandian, Abbottabad, Pakistan 3 College of Pharmacy, King Saud University, 11362 Riyadh, Saudi Arabia 4 Department of Soil Sciences, Pir Mehr Ali Shah ARID Agriculture University, Shamsabad, Murree Road, Rawalpindi, Pakistan Corresponding author: [email protected]

ABSTRACT

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Magnetotactic bacteria provide an interesting example for the biosynthesis of magnetic (Fe3O4 or Fe3S4) nanoparticles, synthesized through a process known as biologically controlled mineralization, resulting in complex monodispersed, andnanostructures with unique magnetic properties. In this work, we report a novel aerobic bacterial strain isolated from sludge of an oil refinery. Microscopic and staining analysis revealed that it was a gram positive rod with the capability to thrive in a medium (9K) supplemented, with Fe2+ ions at an acidic pH (̴ 3.2). The magnetic behaviour of these cells was tested by their alignment towards a permanent magnet, and later on confirmed by magnetometery analysis. The X-ray diffraction studies proved the cellular biosynthesis of magnetite nanoparticles inside the bacteria. This novel, bio-nano-magnet, could pave the way for green synthesis of magnetic nanoparticles to be used in industrial and medical applications such as MRI, magnetic hyperthermia and ferrofluids.

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INTRODUCTION

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Microorganisms have inhabited the earth for billions of years, and their activities are vital strengths forming our planetary surroundings through biogeochemical cycles [1, 2]. Microbial biomineralization specifically takes up natural components, and stores minerals either intracellularly or extracellularly. This is of prodigious interest, as they do not only serve as vital biosignature for searchinghints of past life, but also have numerous commercial applications [3, 4]. Despite the fact, that microbes make an extensive assortment of biominerals, very little is known about these microorganisms’ minerals and their mechanisms of biomineralization [5-7]. The best considered among them are magnetotactic bacteria (MTB), which biomineralize iron oxide (magnetite, Fe3O4) or the iron sulfide (greigite, Fe3S4), inside membrane-bound organelles called magnetosomes (Fig. 1)[8, 9].

Figure 1: A cartoon showing the arrangement of magnetosomes inside magnetotactic bacteria. Magnetosomes consist of a double membrane bound magnetic nanoparticle (MNP) core.

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MTB are widespread, motile, morphologically, phylogenetically, and physiologically diverse group of ubiquitous gram-negative bacteria, which have the ability to orientate, and migrate along magnetic field lines of the earth [10-12]. This capacity of these bacteria relies on the magnetic structures, which are present intracellularly, the magnetosomes. They consist of membrane bound, nano-sized crystals of iron oxide, synthesized by a tightly regulated cellular mechanism, known as Biologically Controlled Mineralization (BCM) [13, 14]. The magnetic crystals of iron are organized into chains by means of a devoted cytoskeleton, and are in charge of the cell magnetotaxis behaviour [10, 15]. The magnetosome synthesis by all accounts is a complicated procedure that comprises a few discrete strides including the formation of a vesicle for magnetosome, uptake of iron by the cell, transport of this iron into the magnetosome vesicle, and biomineralization of Fe3O4 or Fe3S4 in a controlled manner in the cellular nanoreactors [16-18]. Magnetosomes synthesized through BCM by MTB have many interesting attributes, compared with chemically synthesized magnetic nanoparticles. They have high substance purity, limited size extent, specie-specific crystal morphologies, and display specific cellular arrangements of these crystals [19, 20]. These obvious advantages make them convenient bearers for the coupling of relatively higher quantity of bioactive materials, which can later on be alienated by the application of a magnetic field [21]. They can find applications in the recognition of nucleotide polymorphism [3], immunoassays [10], DNA extraction, and pathogens detection in food, in magnetic hyperthermia and (targeted) drug delivery [22].

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MTB are a group of heterogeneous aquatic prokaryotes and due to their high abundance (in marine and freshwater), they play a vital role in numerous sediments, and biogeochemical cycling of iron, as well as other elements [2, 23]. They are mostly microaerophiles or anaerobes, and are attracted toward an environment that contains practically no oxygen [24, 25]. The MTB presence depends on opposing gradient of reducing and oxidizing sulfur species and oxygen. Availability of soluble iron is one of the reasons for their abundance in the particular environment. MTBs are considered as a typical example of gradient organisms [26]. They reside in the first top centimetres of marine sediment, within the oxic/anoxic interface (OAI), where oxygen is present at low concentrations. MTB isolation and culture in laboratory is difficult as they have diverse nutritional requirements, and are redox-sensitive. These conditions are not easy to mimic in laboratory set-ups [27, 28]. MTB that have the capacity to biomineralize under aerobic conditions do exist in our environment, but there is limited published literature available about their isolation, optimization, and culture in lab controlled conditions [29-31]. For instance, Matsunaga et al., isolated helical shaped MTB from fresh water sediments in a carbon medium with ferric gallate [30]. Similarly, Elcey et al., and Jun et al., reported the isolation of MTB capable of thriving under aerobic conditions. They both described the isolation of rod shaped MTB; the former in 9K while the latter in liquid 2219 (ferric quinate) medium [29, 31]. The current study reports an aerobic magnetotactic bacterial strain characterized (bio/chemical and physical) and optimized for the biosynthesis of magnetic nanoparticles. MATERIALS & METHODS Sampling & Culturing Samples were collected from oil refinery sludge in pre-sterilized 50 mL conical tubes, wrapped in aluminium foil to prevent it from direct sun light, and stored in cool/dark place. Each time 2g of sample was dissolved in 50 mL distilled water/ filtered. The samples were

ACCEPTED MANUSCRIPT cultured initially (12 mL) LB broth, and inoculated with 2 µL of sample filtrate and incubated overnight at 30 ºC. The cultured bacterial samples were grown in 9K medium[32], i.e. 15 mL conical tubes were filled with 9K and were inoculated with 100 µL inoculum. The tubes were incubated overnight at 30ºC on continuous shaking (120 rpm) (LSI-100C, KWF China) under aerobic conditions. Bacterial growth was determined at regular intervals of every 2 hr, by recording the optical density (OD) at 600 nm using spectrophotometer (Genesys 10-S, Thermo Spectronic). Magnetic Movement & Gram staining

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Preliminary tests were performed to check if the cultured bacteria are magnetic(magnetotactic) or not. The magnetic movement of bacteria was checked by using a permanent magnet. 5 mL of actively growing bacteria were collected, and centrifuged at 4,000 x g. The pellet was washed in 0.9 M NaCl solution three times. The purified cells were resuspended in 0.9 M normal saline solution. A permanent magnet was applied at one end of a glass tube to align the cells towards the magnetic field gradient. To estimate their relative magnetic behaviour, commercially purchased magnetic nanoparticles were also tested the same way. The Gram staining was performed as per standard procedure. Magnetosomes Extraction

Electron Microscopy Imaging

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The bacterial culture (45 mL) was taken in 50 mL tubes and spun at 12,000 xg (Eppendorf 5810 R, Germany) for 2 min. The pellet was washed thrice with 0.9 M normal saline, and suspended in 0.2% SDS for 45 min at room temperature. The cell-SDS mixture was then sonicated (Cole Parmer CPX 130) at 20 kHz for 5 min at 40% amplitude (pulse on 2 sec, pulse off 1 sec). The sonicated mixture was purified by placing a strong magnet to one side of the tube, followed by removal of fluid containing cellular debris. Extracted magnetosomes were washed with normal saline and stored in PBS buffer at 4 ºC.

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Magnetometry

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Electron microscopy imaging of the bacterial cells, and extracted magnetosomes was performed in Scanning Electron Microscope (SEM) (JOEL (JSM5910, Japan). The sample (5 µL) was poured on Si wafer, dried in air and washed with ultrapure water to remove any salt crystals present in the sample. EDX spectra were collected (at 5 kV) for the samples that were imaged in SEM.

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Magnetometry analysis was performed in a MPMS-VSM (Magnetic Property Measurement System-Vibrating Sample Magnetometer) (Quantum Design, US). Specially designed diamagnetic containers were filled with a dried (powdered) samples, and the M-vs-H was calculated at 300 K. The samples were centred by applying a magnetic field of 1000 Oe. X-Ray Diffraction

Purified magnetosomes from bacterial cells were dried on a clean glass slide, and scraped off onto a zero background Si holder. The phases were identified by XRD in an x-ray diffractometer (X’Pert, PANlytic, Netherlands) in reflection mode. Dynamic Light Scattering DLS was performed to calculate the hydrodynamic diameter of magnetosomes as well as the polydispersity index. A diluted sample of 50 µL was poured in the special cuvette, and it was installed in the particle sizing machine (ZetaSizer ZS Nano, Malvern, UK).

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RESULTS & DISCUSSION Bacterial Cell Culture, Optimization and Biochemical Characterization

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The isolated bacterial sample was cultured in 9K a number of time to reproduce the results. It was found out that the isolated bacterial strain was able to grow in 9K aerobically at 30-32 ºC (pH 3.2), and was always magnetic. The growth plot of bacterial strain (Fig.2A) shows that these cells required 3 hours as their lag phase, followed by active cell division, and rapid increase in the cell number until the 15th hour. This was followed by a 2.5 hours stationary phase, which lead to the decline phase after 22-24 hours. There is a drop observed in the number of bacterial cells with the passage of time (as it can be seen in Fig. 2A), i.e. the cellular densities after 24-28 hrs drastically declined. This could be due the fact that the cells derived their energy from the oxidation of iron ions, and inorganic sulphur compounds [33, 34], which eventually led to the depletion of these minerals along with the accumulation of toxic/waste products. This embarked the decline phase of the growth curve when the toll of cell death overcome cell division by several folds.

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When the growth patterns of the isolated aerobic MTB were compared culturing them in 9K prepared either with Fe2+ or Fe3+ salts, the results were different. It was evident that the cells thrived better in 9K medium with Fe3+[Fe(NO3)3] salts (Fig. 3A). Despite the fact this MTB strain had been isolated in 9K with Fe2+ salts (Fig. 3B), the cellular densities it attained in Fe3+ supplemented growth medium were better than their counterparts. This is consistent with the reported literature where MTB had been cultured mostly with Fe3+ (ferric quinate) salts [35-37]. It is already well-known that ferric ions are reduced to ferrous during the biosynthesis of magnetosomes [38, 39]. Moreover, it was found that the best iron salt concentration with 9K was found to be 4% for Fe2+ and 3% for Fe3+ supplementation.

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The biochemical characterization performed for our isolated aerobic MTB is given in table 1.The molecular identification of the novel isolated magnetotactic bacterial strain has been performed through 16S rRNA sequencing via PCR amplification, and discussed in detailed in references [40, 41]. Magnetic Movement & Gram Staining

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Gram staining of the bacterial strain revealed that they are novel gram positive rods (Fig. 2B). This is contrary to all of the reported literature which shows that MTB are gram negative [10, 28, 42]. The cultured bacterial cells upon application of permanent magnet (externally) aligned parallel towards the field gradient. It was observed in this experimental test even by naked eye that these cells did harbour magnetic content inside them which pushed them to swim towards the magnetic field gradient and aligned towards it. When the same procedure was repeated with only 9K medium (as a negative control), there was no movement observed. Moreover, the same experiment was repeated with commercially synthesized iron oxide nanoparticles. The movement of these chemically synthesized nanoparticles was more rapid than our cells (Fig. 2C). A possible reason could be the fact that commercially synthesized nanoparticles were free of any attached proteins, while bacterial magnetic nanoparticles were wrapped around in a number of sheaths of proteins, and lipids in a cellular environment [6, 21]. This made them move relatively slower towards the permanent magnet. This also pointed towards the fact that the yield (and the) rate at which

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the magnetosomes were biosynthesized would be relatively slower/lower. Our reported aerobic bacterial strain was cultured in a relatively simple growth medium that lacked any Csource or vitamins. The absence of these important organic substances affected the yield of biosynthesized magnetosomes, as well as the cellular densities recorded during their aerobic growth plot (Fig. 2A). Most of the reports of culturing MTB for harvesting magnetic nanoparticles were rather cultured in growth media, and well supplemented with organic compounds that boost cellular thriving [43, 44].

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Colony Morphology Negative Rod Large Undulate Raised Wavy Creamy white Dry Staining Tests Gram staining Positive Capsule staining -----Spore staining Positive Biochemical Tests Oxidase test Negative Citrate test Positive Coagulase test Positive Urease test Negative Catalase test Positive Nitrate reduction test Positive Indole Negative Motility test Positive Methyl red test Negative Voges proskauer test Negative Triple Sugar Iron Test Glucose Positive Lactose Negative Sucrose Negative H2S gas production Negative Hemolysis test Positive (beta –hemolysis) Table 1. Biochemical Characterization (Tests) for isolated MTB

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Pigment Shape Size Margins Elevations Surface Color Texture

Electron Microscopy Scanning electron microscopy revealed that these bacterial rods are roughly 4-5 μm in length and ̴2 μm in diameter (Fig. 2D). It can be seen (Fig. 2F) that the magnetite nanoparticles resembled the cubo-octahedral shape. The STEM image shows that there were small distances between them. They appear to be not connected to each other, and gave an impression that they were just lying next to each other. This small gap indeed showed the proteins wrapped around each mineralized nanoparticle, and keeping them in chain like

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structures, the magnetosomes, but we could not see them clearly under a STEM. EDX spectrum collected for magnetosomes isolated from bacterial cells shows clear iron (Fe) peak easily detected in EDX spectrum (Fig. 2E). A very sharp peak for Si is also visible and this originated from the substrate since the imaging was performed on a Si wafer. All other peaks were coming from the sample holder (or stage) of the microscope.

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Figure 2: A) Growth plot of isolated MTB strain in 9K at 30 ºC (B) Isolated MTB strain: a gram positive Bacillus (C) Magnetic response towards a permanent magnetic; isolated MTB cells slowly aligned towards the magnetic field gradient as compared to commercially synthesized magnetic nanoparticles (D) SEM image of isolated MTB rods (E) EDX spectrum of extracted magnetosomes showing clear peak of Fe (F) Scanning Transmission Electron Microscopy image of isolated magnetosomes

Figure 3: Growth plots of isolated MTB in 9K with 3%, 4% and 5% salts of (A) Fe 2+ and (B) Fe3+

Dynamic Light Scattering (DLS) The aim in this experimental test was to determine the size (diameter) of individual magnetite nanoparticles, rather than the size of chain(s) of magnetosomes. DLS data shows that the sample of extracted magnetosomes was relatively monodispersed, as it yielded a low PdI (Polydispersity Index) value (Fig. 4A). The correlation coefficient shows that the signal decayed just after 100 μs (Fig. 4B), thus again proving the fact that the sample was monodispersed, and free from micrometre sized particles. The hydrodynamic diameter found

ACCEPTED MANUSCRIPT for magnetosomes particle was around 34.6 nm. There is indeed difference of size between values obtained from microscopy and DLS. The reason could be ultrasonication of magnetosome particles at a higher amplitude, and prolonged duration for samples prepared for DLS that had detached the nanoparticles from the magnetosome chains.

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Z-Average (d/nm): 34.60 PdI: 0.232

X-Ray Diffraction

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Figure 4. DLS of magnetosomes. (A) Size distribution by intensity showing a distinctive peakat 34 nm and lacking any other peaks in the sample. (B) Raw correlation data showing the signal decay shortly after 100 μs

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The XRD analysis was performed in the first phase with relatively lower amounts of purified magnetosomes. The low quantity of purified magnetosomes XRD pattern had a lot of noise, and only one characteristic peak for magnetite was identified (Fig. 5A). However, when the amount was increased, a several folds higher intensity signal was recorded as evident in the XRD pattern (Fig. 5B). The results shown proved that sample was not only magnetite but was also crystalline.. Close inspection of the pattern showed that several peaks (220), (311), (400), (511) are indeed the characteristic magnetite peaks as published by Sachin et al. [45] recently. Moreover, we also obtained strong signals fitting to salts (NaCl) that could potentially be left behind in the sample as impurities (NaCl was the solution in which the cells were resuspended after several cycles of washing).The obtained XRD data had ruled out the possibility of presence of greigite in isolated magnetosomes.

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Figure 5. XRD pattern of magnetosomes; (A) obtained with relatively lower amount of magnetosomes and (B) pattern with higher amount.

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Magnetometry

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Physical characterization of the extracted magnetosomes was performed in a VSM, i.e. magnetization was plotted as a function of the applied magnetic field at room temperature. The results showed (Fig. 6) that these particles were indeed magnetic yielding a saturation of magnetization at 3000 Oe magnetic field. The maximum magnetic moment recorded for these magnetosomes was 0.40 emu/g. It was difficult to confirm, if these particles were superparamagnetic or ferromagnetic, because the plot had indeed a small amount of remanence, and coercivity recorded in the hysteresis loop. Therefore, it is concluded that these particles were ferromagnetic in nature with very low remanence and coercive fields.

Figure 6. Magnetization as function of applied magnetic field for purified magnetosomes.

Conclusion In conclusion, we report here a magnetotactic bacteria capable of producing crystalline magnetic (magnetite) nanoparticles intracellularly under aerobic conditions in a chemically simple medium. These magnetic nanoparticles can be isolated, purified and characterized in terms of their physical and chemical properties as an alternative to replace their chemically synthesized counterparts.

ACCEPTED MANUSCRIPT Acknowledgements This work was supported by COMSATS IIT Research Grant Program, “The Living NanoMagnets” (No. 16-62/CRGP/CIIT/IBD/15/790). The authors highly appreciate the help of Dr. Sadia Sattar, Dr. Maira Janjua, Dr. Fareeha Siddiqui, and Dr. Sundus Javed for assistance with some of the experimental work. We highly appreciate the help of Mr. Maahil Arshad and Miss Palwasha Ishtiaq (native English speakers) in correcting the overall language (flow) and punctuation of the paper. References

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GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT HIGHLIGHTS “Respiring Cellular Nano-Magnets” Ref: MSEC_2016_193_R2

The authors are indeed grateful to the editor and the Reviewer(s) for thoroughly reading our paper and advising revisions to make our work good enough to be published. We have taken the comments of

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Reviewer 3 very seriously and have responded to them and accordingly modified our manuscript to the best of our capabilities in the following ways

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a. The English language has been corrected with help of native speakers all the punctuation and overall flow of the paper had been improved.

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b. The previous works published on aerobic MTB had been incorporated into the manuscript. c. All of the previous reports about magnetotactic bacteria show that MTB are gram negative while our

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novel strain is a gram positive. This has been highlighted in the manuscript along with necessary references/citations.

d. The magnetite peaks obtained through XRD were compared with out previously reported paper and we

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found them similar. In this regard, we have cited one of our published articles to confirm these peaks are characteristic peaks of magnetite as magnetic nanoparticles biosynthesized by MTB.

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e. The 16S RNA PCR mediated identification and Phylogenetics has been performed already and has

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been submitted in another ELSVIER journal for publication. We have cited the unpublished paper as well as the thesis of the student who performed this. This is to mention that we had sought the permission of the editor for this revision to be done as such.

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This is to state that we have responded to all of the comments of the reviewers/EIC and has revised our manuscript accordingly. We hope that our manuscript now will be good enough to be accepted for

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publication in your esteem journal. However, if there is still any short-comings, our team will gladly incorporate more changes. Sincerely,

Abid Ali Khan