Digital printing of cyanobacteria

The model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis)20, 21 was chosen for our studies as it has been used extensively in BPV devices22 and is amenable to metabolic engineering12. To help minimise clogging or damage to Synechocystis cells during printing, we decided to test a Hewlett-Packard (HP) Deskjet 340 inkjet printer which contains an ink cartridge with a 50 µm wide nozzle, one of the largest available commercially. In comparison, the average diameter of a typical coccoid Synechocystis cell is about 1.5 µm23. The thermal inkjet technology used by the HP Deskjet printer is also more benign for cell printing than piezoelectric inkjet technology16, 24

Initial experiments revealed that Synechocystis cells printed onto ordinary paper could be grown on top of an agar plate (Fig. 1a). Paper is increasingly considered as an attractive candidate for the development of disposable electronics due to the advantages of low cost, widespread availability, flexibility and environmental friendliness2, 25. Analysis of chlorophyll fluorescence using an imaging Pulse Amplitude Modulated fluorometer (PAM) confirmed that the incubated cells were photosynthetically competent. The maximum quantum efficiency of photosystem II (PSII), determined from the ratio of variable (Fv) to maximum (Fm) chlorophyll fluorescence (Fv/Fm), measured using single saturating light pulses, was found to be about 0.4 (Fig. 1b), in good agreement with values measured for cyanobacteria in liquid cultures (Fv/Fm = 0.3–0.5)26, 27. The printing process did not affect cell viability based on a comparison of the number of colony forming units before and after printing (Fig. 1c; Supplementary Table 1). The chlorophyll concentration of the printed cells on paper after the incubation was approximately 50 µg cm−2, which is similar to that of a plant leaf28.

Fig. 1 Cell viability and photosynthetic capabilities of digitally printed cyanobacteria. a Photograph of inkjet-printed Synechocystis cells after 3 days of incubation. Scale bar measures 2 cm. b Chlorophyll fluorescence image of the sample a by imaging PAM, showing maximum quantum efficiency of PSII (Fv/Fm) at the values of about 0.4 according to colour gradient in the legend bar. c The panel compares the growth of Synechocystis colonies before and after the inkjet printing process, following 5 days of incubation on a BG-11 agar plate. A 3 µl aliquot of cells from a dilution series representing 10−1, 10−2 and 10−3 of the original suspension was spotted. For the most dilute cell suspension taken after printing, 90.5 ± 10.6 colonies were counted, whereas 87.5 ± 12.0 colonies were counted before printing. The difference between these values was found to be not statistically significant (one-way ANOVA: p = 0.815) (Supplementary Table 1) Full size image

Growth of printed Synechocystis cells on other porous (edible rice paper, nano-paper, woven fabric) and non-porous supports (inkjet coated plastic, indium tin oxide coated polyethylene terephthalate (ITO-PET)) was much poorer than on paper (Supplementary Figs. 1, 2), probably because these materials lack the microporous structure of paper required for high water absorption and the fibrous matrix needed for efficient wicking25.

An alternative methodology based on pneumatic microvalve inkjet printers adopted in cell printing for biofabrication18, was also tested (Supplementary Fig. 3). However, the larger volumes dispensed by this system led to over-wetting of the paper substrate, which hindered the precise and uniform deposition of the cyanobacterial cells on the paper support.

Construction and characterisation of a digitally printed bioelectrode

To test the electrogenic properties of printed cyanobacteria, we fabricated a bioelectrode, which is defined as the combination of photosynthetic organisms with an inert electrode material29. The cyanobacterial bioelectrode was printed in a two-step process: firstly, the electrode was printed on the paper substrate using an inorganic conductive inkjet ink and secondly the cyanobacteria were printed onto the electrode pattern on the paper. The conductive inkjet ink we chose was the “Nink-1000: multiwall” (NanoLab, USA), which consists of carbon nanotubes (CNTs) in aqueous suspension. CNTs have previously been used as conductive patterns on paper substrates30, 31 and have been shown to be compatible with the growth and electrochemical analysis of cyanobacteria32. We found that 5 to 6 overlays of the Nink-1000 conductive ink could be printed to give a conductive surface with a resistivity in the range of 5–10 kΩ cm and that cyanobacteria could be printed and grown directly on the CNT electrode on paper (data not shown).

In order to compare the performance of our printed bioelectrode to the bioelectrode formed by gravity-deposition of cells22, characterisation was carried out by forming a ‘hybrid’ biophotovoltaic cell consisting of the printed bioelectrode paired with the platinised carbon cathode electrode used in conventional BPV devices. Figure 2a–c illustrates the assembled hybrid BPV cell in which the cathode is exposed to the air as in the conventional BPV system.

Fig. 2 Electrochemical characterisation of a digitally printed bioanode in a hybrid BPV system. a Schematic representation (semi-exploded view) of the BPV unit with printed paper-based anode. Clamping screws (component 1); marine grade stainless steel ring for contacting the CNT anode (component 2); printed CNT anode in black (Ø 60 mm) with printed photosynthetic organisms in green (Ø 40 mm) with a total area of ~ 28.4 cm2 (component 3); hydrogel (component 4); Plexiglas vessel (component 5); carbon paper-Pt, with a total area of ~ 3.5 cm2 was used as cathode (component 6); silicon O-ring (component 7); stainless steel plate used to clamp all the component together (component 8). ~ 60 ml of BG-11 medium was placed above the printed cells in the chamber formed by the top plate. b Schematic representation of the BPV unit cross-section where electrons, protons and oxygen flow are also shown. Numbering as in a. c Photograph of the experimental setup (excluding the potentiostat and the wiring). Numbering as in a. d Polarization and e power curves for the printed anode in the BPV unit. Printed Synechocystis (incubated for 5 days after printing) on printed CNT anode exposed to light (magenta symbols) and in the dark (grey symbol) was compared with a bare printed anode (black trace). Number of repeats is indicated in parenthesis Full size image

Polarisation curves (Fig. 2d) were used to characterize the printed Synechocystis bioelectrode and were recorded by performing linear sweep voltammetries (LS) in the absence and in the presence of light (100 µE m−2 s−1). The maximum current density output generated by the printed cells was found to be just over 4 mA m−2 in the light and 3 mA m−2 in the dark (Fig. 2d). This range is approximately 3 to 4-fold higher than previously recorded (ca. 1 mA m−2) for the same Synechocystis strain deposited on an ITO-PET electrode using the conventional approach22.

Power curves (Fig. 2e), derived from the polarisation curves using Ohm’s law, showed a clear effect of light with a peak power output of 0.38 ± 0.07 mWm−2 and 0.22 ± 0.07 mWm−2 in the light and in the dark, respectively, a difference that was found to be statistically significant (one-way ANOVA: p < 0.0005) (Supplementary Table 2). This range is again 3 to 4-fold higher than previously recorded (ca. 0.12 mW m−2 with a slight difference between the dark and light cycles)22.

In the absence of cells, the peak power output was considerably lower (0.07 ± 0.01 mW m−2; Fig. 2e, black symbols) and insensitive to the presence of light (data not shown). The difference in power output in the dark with and without the cells on the anode was found to be statistically significant (one-way ANOVA: p = 0.005) (Supplementary Table 3).

The printed system was characterised further by chronoamperometry, which monitors the current output as a function of time and records changes induced by external stimuli such as light. The chronoamperometric experiments were performed at three different light intensities (100, 250 and 500 µE m−2 s−1) separated by periods of 1 h in the dark. Increases in current output were observed only in the samples with the printed cyanobacteria while no changes were observed in the controls (Fig. 3a). As shown in Fig. 3a the currents measured in the presence of light were higher than in the dark and their magnitudes were comparable to the ones measured in the potential scanning experiments (Fig. 2d). Figure 3b shows the values of the total charge accumulated as a function of the intensity of the light, calculated by integrating the current output over time and subtracting the contribution of the dark current. The device exhibits the expected light saturation of the photosynthetic apparatus12 with saturation of the current output observed at light intensities above 200 µE m−2 s−1.

Fig. 3 Effect of light intensity on anodic photocurrent produced by the hybrid BPV system. a Current output measured over 6 h with three periods of light and darkness (1 h each). Light periods indicated by the yellow bars. Black trace for inkjet-printed Synechocystis on printed CNT anode and magenta trace for control experiments without the printed cells. Number of repeats is indicated in parenthesis. b Saturation curve for the current outputs as presented in the a. For each period of light (100, 250 and 500 µE m−2 s−1), the current was integrated over time. The charge attributable to dark current over the same time was subtracted from the total charge during the light periods and plotted vs. the photon flux. Each data point is the result of 9 replicates and the standard error is shown as error bars Full size image

Powering a digital clock

To assess the ability of the printed bioelectrode to power a small electronic device, such as a biosensor, we tested whether the hybrid BPV unit shown in Fig. 2 could power a digital clock. This test allows direct comparison with literature reports where conventional BPV devices have been shown to power a digital clock when connected in series22.

Nine replicates of the hybrid BPV unit were arranged in three clusters connected in parallel. Each cluster had three units connected in series (Fig. 4a). This setup produced an overall voltage output of 1.4–1.5 V and an overall current output of 1.5–2 µA, a good compromise based on the clock manufacturer’s specifications for powering the digital clock.

Fig. 4 Powering a clock and a LED-flash with an array of Hybrid BPV units. a Schematic representation of the experimental setup for the powering of a digital clock. An array consisting of 9 Hybrid BPV cells were organised in 3 clusters connected in parallel. Each cluster had 3 units connected in series. b Chronovoltammetric and chronoamperometric traces recorded during the experiment where the circuit (i.e., the digital clock) was either on (i.e., clock activated) or off (i.e., clock deactivated) for periods of approximately 30 min. c Schematic representation of the experimental setup for the powering of a LED. The array was organised all in series. d Chronovoltammetric and chronoamperometric traces recorded during the experiment where the circuit with its integrated LED was either on (i.e., pulsing every 2.5 s to activate the LED) for periods of approximately 60 s or off (i.e., LED deactivated) for periods of approximately 1 h. Rate of voltage recovery was estimated by fitting the last 7 s of data with a linear regression line (in red); e kinetics of recovering to the original voltage following LED pulse when the BPV array was kept in the dark and when it was exposed to light; f average energy consumed for each LED pulse Full size image

We found that the digital clock was successfully powered by the BPV array for ‘ON’ periods of 30 min alternated with ‘OFF’ intervals of 30 min to allow the BPV devices to recover (Fig. 4b). The chronovoltammetry and chronoamperometry curves in Fig. 4b clearly indicate the discharge of the array when connected to the clock to activate it (‘ON’): there was a rapid decrease followed by stabilisation of the voltage. On disconnection, there was a rapid increase in voltage followed by stabilisation of the potential across the anode and cathode. This process was repeated several times demonstrating reproducibility.

Powering a LED

Small and low power electronic devices such as biosensors often work over short measuring periods, interspaced by longer periods of inactivity. To assess the ability to generate a relatively high power output in short bursts, we tested whether Hybrid BPV units could generate flashes of light from an LED (Fig. 4c).

The LED was connected to a pulse generator whose electronic scheme is presented in the supporting information (Supplementary Fig. 4). To generate the required voltage (ca. 3 V), an array consisting of 9 Hybrid BPV cells was connected in series (Fig. 4c), so that the output voltage is the sum of the 9 units. To accumulate the required charge, the BPV array was charged for 1 h, then the circuit was closed for 60 s during which the LED was pulsed at a frequency of one pulse every 2.5 s. We detected in ten separate experiments an average of 24 flashes in this 60 s period, confirming that the BPV array could indeed generate bursts of power sufficient to drive the LED (Fig. 4d).

Immediately after closing the circuit a short spike of current intake (~ 35 µA) and potential drop (~ 1.5 V) lasting ~ 2 s were observed (Fig. 4d). This vigorous initial electrical consumption is an expected phenomenon due the circuit capacitance and a similar behaviour was also observed with the digital clock (Fig. 4b). For the remaining 58 s, the current driven by the board stabilised at around 2–3 µA with a closed-circuit potential of ~ 2 V.

Recovery of the voltage following the LED discharge was also monitored and its kinetics were measured when the BPV array was kept in the dark and when it was exposed to light. Figure 4e shows that in the presence of light (100 µE m−2 s−1) the recovery was faster suggesting that the photosynthetic reactions in the cyanobacteria accelerate the recovery of the system following discharge. Fig. 4f shows the energy consumption at each flash of the LED light. The dark/light regime did not cause any significant variation in the energy consumption. As expected, the energy delivered by the pulse generator was independent of the activity of the BPV array, as the energy (~ 13 µJ) was delivered to the LED only when the capacitor of the pulse generator was charged.

Design and testing of a semi-dry thin-film BPV system

Having demonstrated the ability of the printed bioelectrode (as a bioanode) to generate a sustained power output in a hybrid BPV system, we fabricated a printed BPV cell where not only the anode but also the cathode was patterned and printed on paper. Power output in microbial fuel cells strongly depends on the overall dimensions of the electrodes and the relative distance between the planes of the anode and the cathode5, 33. In order to maintain a compact size, we designed a zigzag electrode pattern with a view to enabling the conduction of ions between anode and cathode while reducing the overall length of the electrodes. Furthermore, the cathode surface area was made larger than that of the anode in order to enhance the exposure to oxygen and reduce catalytic limitations5, 33.

As before, a two-step inkjet printing process was used to make the printed BPV system with the electrogenic cyanobacteria deposited precisely on the zigzag anode (Fig. 5). Our tests confirmed that 5 to 6 overlays of the ink printed gave a conductive surface with a resistivity of the order of 100 kΩ cm. This value is higher than that observed previously (Fig. 2) due to variability between different batches of the Nink-1000 ink. Nevertheless, Synechocystis (data not shown) as well as the related cyanobacterium Synechococcus sp. PCC 7002 could be printed and grown directly on the conductive ink (Fig. 5).

Fig. 5 Design of a fully printed BPV system. a Schematic representation (semi-exploded view) of the digitally printed bioelectrode module. 1: Printed photosynthetic organisms in green; 2: Printed CNT anode; 3: Printed CNT cathode; 4: Paper substrate. The one module consists of one zigzag anode and one zigzag cathode with surface areas 1.36 cm2 and 2.73 cm2, respectively. b Photograph of A4-size arrays with freshly printed Synechococcus cells, compared to the incubated module grown on an agar plate for 3 days. (Note the enhanced green colour of the growing cyanobacteria.) 1–4 are the same as a and 5 is the solid medium Full size image

The printed bioelectrode module made with Synechocystis cells was assembled into a thin film configuration by covering the anode and cathode with a water-absorbent gel (hydrogel) so as to form a slim BPV construct. Hydrogels have previously been used to encapsulate microorganisms in MFCs34, 35, but in our application the hydrogel covering the biofilm functions both as a salt bridge connecting the anode and cathode and as a supply of minimal growth medium and water to the printed cells (Fig. 6). It plays an equivalent role to the bulky liquid reservoir in conventional BPV systems and, together with the paper-based solid culture system, reduces the volume of the BPV device substantially. As indicated in the Methods section, the assembled system was then placed into a chamber where humidity and illumination were controlled to prevent dehydration.

Fig. 6 Testing the performance of the fully printed BPV device. a Schematic representation (semi-exploded view) of the printed paper-based BPV cell. Paper support in light grey (component 5); Printed CNT anode (component 3) and CNT cathode (component 4) in black; Printed Synechocystis in green (component 2); Bridging hydrogel in pale blue (component 1). b Photograph of the experimental setup (excluding the potentiostat), showing a pair of BPV modules printed in series. c Schematic representation of the BPV cross-section where electron, proton and oxygen flows are also shown. d Power output measured over 4 days with periods of light and darkness. Light periods indicated by the yellow bars. Magenta trace for inkjet-printed Synechocystis on printed CNT anode and black trace for control experiments without the cells Full size image

As this configuration was difficult to connect to an external reference electrode, the electrochemical characterisation was carried out with a voltmeter and an external load33. The recorded power output (Fig. 6d) was found to be smaller than that measured in either the hybrid BPV system of Fig. 2 or the literature values of a conventional BPV system21. The reduction could be due to an increase in the internal resistance of the printed circuit since the magnitude of the decrease in the power output matched the increase in the ink resistivity

Long-term stability of the power output was assessed over a 10 h light/14 h dark cycle for 4 days (Fig. 6d). During each light/dark cycle, the peak power reached a maximum within two to three hours and then remained stable until the light source was switched off, after which the power returned to the stable value recorded in the dark. Overall, power output was observed to be stable (within a 10 % error) during the light/dark cycles throughout the period of over 100 h.