Bacterial Nanobionics via 3D Printing

Transcription

1 Supporting Information Bacterial Nanobionics via 3D Printing Sudeep Joshi, Ellexis Cook, and Manu S. Mannoor* Culturing of cyanobacteria. Fresh cyanobacterial cultures (151710) were procured from Carolina Biological, NC, USA and was grown in an environmental chamber with controlled temperature, humidity, light intensity, and air ventilation. The growing cyanobacteria culture was subjected to temperature of ~ 25 ± 2 ºC with 12-hours of light period using fluorescent lamps (70 foot-candles of fluorescent light from the ceiling) followed by another 12-hours period of darkness. The subcultures were prepared by pipetting 200 µl of inoculum for every 20 ml of Alga-Gro freshwater medium (ph ~ 7.8), (Carolina Scientific ) into a sterile polycarbonate container. The cyanobacterial cell growth was periodically monitored by measuring the optical density (OD) of the growing culture using a UV-visible spectrophotometer (Cole-Parmer). Preparation of printing inks (Bio-ink and Electronic-ink). To constitute cyanobacterial bio-ink, pharm grade Alginate LF 200 FTS (FMC Corporation, PA) was procured and used in our experiments without any further modifications. Freshly growing cyanobacterial cell culture in nutrient-rich medium (Alga-Gro freshwater media 1

2 by Carolina), was gently mixed with Alginate and vortexed lightly to achieve a uniform cyanobacterial cell suspended bio-ink. In the present experimental work, different concentrations of bio-ink (1% to 5.5% w/v, with an interval of 1.5%) were tested to achieve optimal concentration of 4% w/v for 3D printing purpose. The cross-linking of the 3D printed alginate-based bio-ink structure was initiated by periodic spraying of CaCl2 (0.25 M concentration in DI water) with nozzle spray on to extruded 3D structures. Preparation of electronic-ink constituting GNRs uniformly-dispersed in PEDOT: PSS is described in experimental methods. Figure S1. shows the rheological studies, wherein viscosity as a function of shear rate variation indicates shear-thinning behavior, which signifies uniformly-dispersed suspension of as-prepared printing inks. Figure S1. Rheological characteristics of inks used for printing. (A) Variation of viscosity as a function of shear rate for bio-ink 4% (w/v), and (B) electronic-ink 2% (w/v). 2

3 Optimization of bio-ink concentration. Figure S2 shows 3D printed structures with different concentrations of bio-ink used for the optimization process. It is evidently visible from figures that lower concentrations of bio-ink have lesser viscosity and the printed structure doesn t hold the prescribed shape. At lower concentrations of 2% (w/v), the dumble-effect is pronounced leading towards distorted geometry (Figure S2B). The optimized concentration of 4% (w/v) resulted in well-defined prescribed geometry and the extruded structure does hold the desired shape and there was no visible dumbleeffect present (Figure S2C). To showcase the versatility of the optimized bio-ink, self-standing structures were 3D printed with height of about 7-8 mm (Figure 2E). Remarkably, these 3D printed samples exhibit anisotropic arrangement of cyanobacterial cells leading towards densely-packed geometry in the direction of prescribed printing path. 3

4 Figure S2. (A - C) Bio-ink (cyanobacterial cell suspension within alginate hydrogel matrix) was 3D printed in different geometries (Scale bar cm). (D - F) Spatial arrangement of cyanobacterial cells emitting auto-fluorescence from the indicated location demonstrates direction-dependent anisotropic cyanobacterial alignment (Scale bar - 20 µm). Photosynthetic bioelectricity generation (Amperometric, current versus time (i-t) curve). Figure S3 demonstrates a detailed schematic illustration of electrode extended through the biotic mushroom s stem for the electrical connection purpose to perform photosynthetic bioelectricity generation studies. As can be seen from the schematic figure, the 3D printed electrode network on mushroom s pileus acts as a working electrode, a platinum wire acts as a counter electrode, and Ag/AgCl acts as a reference electrode, all three immersed in an electrolytic solution. A 100 mm phosphate buffer saline with ph 7.4 was used as the electrolyte solution. The observed photocurrent measurements were performed in between working electrode and counter electrode. Amperometric studies (i-t curve) were recorded using a Keithley source measure unit (SMU) (Model 2450). The light source used for illumination was procured from Dolan-Jenner industries, having variable luminous flux settings. 4

5 Figure S3. Schematic cross-sectional view of the printed electrode network connections inside the biotic mushroom structure and experimental set-up used for photosynthetic bioelectricity generation studies. Control experiment with dead cyanobacterial cells. We have conducted an additional control experiment with dead cyanobacterial cells to support the claim of photocurrent generation resulting from the extracellular electron transport associated with cyanobacterial metabolism. To kill cyanobacterial cells, we have treated these cells with 100% methanol solution and keeping it in dark at 4 C for the duration of 12 hours. Figure S4 shows bright-field and fluorescence microscopic studies conducted to confirm cyanobacterial cell-death from methanol treatment. The cyanobacterial cells treated with 100% methanol solution emitted no red auto-fluorescence confirming cell death. 1 5

6 Figure S4: Microscopic studies for determining live and dead cyanobacterial cells (A and B) Bright-field and fluorescence microscopic images of live cyanobacterial cells exhibiting autofluorescence, respectively. (C and D) Bright-field and fluorescence microscopic images of methanol-killed dead cyanobacterial cells shows no auto-fluorescence, respectively (Scale bar: 50 µm). These dead cyanobacterial cells were then examined for photocurrent generation studies by maintaining all other parameters constant as for healthy/live cyanobacterial cells. As can be noted that, light on/off experiments with dead cyanobacterial cell sample doesn t produce any detectable photocurrent during several light and dark cycles during experiment conducted for the duration of about 45 mins (Figure S5). Therefore, the two control studies (Control 1: Only PEDOT: PSS without cyanobacterial cells (Figure 3G, in manuscript) and Control 2: dead cyanobacterial cells (Figure S5, below)) supports the claim of detected photocurrent generation from the extracellular electron transport associated with cyanobacterial cells during the light illumination studies in presence of a photosynthetically active light source. 6

7 Figure S5: Photocurrent response from control sample (dead cyanobacterial cells) doesn t show any detectable photocurrent during several light on/off cycles conducted for the duration of about 45 mins. Mushroom s structure pertinent for bio-inspiration. Mushroom is evolutionary tailored and possesses unique structural properties that are relevant for bio-inspiration. We conducted scanning electron microscopic (SEM) image analysis of different mushroom parts that reveals distinctive characteristics details of individual part. The pileus of mushroom consists of porous structure of abundant fibrous stripes. Such a porous structure possesses larger surface area, which provides better moisture absorption resulting in mimicking bio-physiological environment for longer survival of cyanobacterial cells leading towards engineered bionic symbiosis demonstrated in our present study. The stem of mushroom 7

8 consists of well-networked fibrous channels that can be effectively used for transporting water molecules via capillary mechanism. Figure S6 shows SEM images depicting structural details of two different sections of a button mushroom. Figure S6. Suitability of mushroom s structure for bio-inspiration. (A) Pileus of mushroom possesses fibrous structure which are porous hence provides larger surface area for efficiently absorbing nutrient-rich medium (Alga-Gro fresh water medium) from 3D printed bio-ink, and acts as a reservoir of essential nutrients for cyanobacterial growth for longer time duration (scale bar - 10 µm). (B) Stem of mushroom consists of channeling architecture suitable for delivering water molecules through capillary action (Scale bar - 10 µm). 8

9 Engineered bionic symbiosis studies with Dead mushroom as a suitable control. We have conducted an additional experimental study with dead mushroom as a suitable biologically relevant control to strengthen the discussion on engineered bionic symbiosis. A comparative study was undertaken to examine the life-span of immobilized cyanobacterial cells on the pileus of live and dead mushroom. In this study, a freshly plucked live mushroom (figure S7A) and a dead mushroom (figure S7B) is used as substrates to immobilized 3D printed cyanobacteria. We have used vinegar solution (20% (v/v)) a natural fungicide due to its high acetic acid content, to kill the mushroom. The pileus of live mushroom was slimy, soft, adipose, and moisture-rich whereas that of dead mushroom was dry and rough. The cyanobacterial cells were 3D printed on pileus of live and dead mushroom for examining the substrate effect on life-span of cyanobacterial cells (figure S7C & S7D). 9

10 Figure S7. (A & B) Live button mushroom and dead button mushroom (vinegar-killed) for comparative studies. (C & D) 3D printed cyanobacterial bio-ink on live and dead mushrooms, respectively. For establishing the claim of engineered bionic symbiosis similar procedure was followed as described for the biotic mushroom (BM) and abiotic mushroom (AM) in the manuscript. Interestingly, cyanobacterial samples collected from live mushroom (figure S8B) shows relatively higher absorbance as compared to samples collected from dead mushroom (figure S8C) for similar collection time (Tc). This observation indicates that live mushroom pileus supports cyanobacterial cell viability for longer time duration, as compared to the dead mushroom. The live mushroom s pileus is rich in moisture content and possesses fibrous stripes which efficiently absorbs the nutrient-rich medium from 3D printed bio-ink, and hence acts as a reservoir of essential nutrients for cyanobacterial cell growth for longer time duration. As can be seen the dead mushroom shrank in dimensions due to moisture loss and significantly differs in texture of pileus and stem as compared to live mushroom, as a result it doesn t provide supporting bio-physiological conditions for cyanobacterial cell viability. Simultaneously, results from the standard plate counting method followed an exactly similar trend, wherein cyanobacterial cells collected during early hours resulted in greater number of isogenic colonies. As evident from the figure S8D, colony count significantly decreased for cyanobacterial samples collected from dead mushroom after Day 1. Therein lies a strong corroboration between results observed with UV-visible spectroscopy and standard plate counting method, which paved the way for existence of engineered bionic symbiosis resulting in the increased life-span of cyanobacterial cells on live mushroom s pileus. Experiments were repeated 10

11 for 5 times during both methods resulting in better statistical claim for establishing the existence of an engineered bionic symbiosis. Figure S8. Engineered bionic symbiosis studies. (A) Photograph of samples showing an evident decrease in cyanobacterial cell density collected from live mushroom and dead mushroom on 3 different days. (B & C) Absorbance spectra variation of (Chl-a) pigment extracted from cyanobacterial samples collected from live mushroom and dead mushroom, respectively. (D) 11

12 Variation of normalized percentage of CFU (Colony Forming Unit) with respect to cyanobacterial samples collected at different time intervals (Tc) from agar plate counting method (N = 5) (Bar colors: Green cyanobacteria samples collected from live mushroom, Red - cyanobacteria samples collected from dead mushroom). References: 1. Liu, B. R.; Huang, Y.-W.; Lee, H.-J. BMC Microbiology 2013, 13, (1),