Assembly of RP/CdS bio-nano-hybrid and characterization
CdS is a commonly used nanoparticle for bio-nano-hybrid cells construction due to its suitable band gap for visible light response (Cheng et al. 2018). Thus, it is speculated to use CdS QDs to assemble the bio-nano-hybrid cells. First of all, the growth curve of the genetic engineered RP strain was characterized. It was found that this engineered RP strain showed typical growth curve with an exponential growth phase (0–96 h) and a steady growth phase (96–192 h) (Additional file 1: Fig. S1). The highest cell density of RP cell could reach an OD660 of ~ 0.35. The CdS QDs synthesized was also characterized by UV–vis and fluorescence analyses, which showed typical spectra of CdS nanoparticles (Additional file 1: Fig. S2). Next, the RP/CdS bio-nano-hybrid cells were constructed by incubating the RP cells with CdS QDs. The CdS QDs were anchored onto the cell surface due to electrostatic interaction. After that, the RP/CdS hybrid cells were isolated and the excessive CdS QDs in the suspension was washed (Fig. 1a). According to the TEM observation (Fig. 1b), it was clear that the RP/CdS hybrid cell consists of RP cell and nanoparticles. Many nanoparticles usually showed biotoxicity, which might be detrimental to microorganisms (Zhu et al. 2019). Thus, the cell viability of the RP/CdS hybrid was determined by using LIVE-DEAD Baclight staining and colony formation unit (CFU) analysis. As shown in Fig. 1c, the RP/CdS hybrid cells showed dominant green fluorescence, indicating high cell viability. According to the image analysis and statistical calculation, the cell viability of RP/CdS hybrid cells was about 98.9 ± 0.5%. Moreover, the CFU analysis indicated there was no statistically significant difference (p > 0.1) between bare RP cells and RP/CdS hybrid cells (Fig. 1d). Thus, these results suggested the RP/CdS bio-nano-hybrid cells with high cell viability were successfully constructed.
To further confirm the formation of the RP/CdS bio-nano-hybrid cells, XRD, Raman and X-ray energy-dispersive spectroscopy (EDS) analyses were performed. It was observed that the pure CdS QDs showed typical diffraction peaks that could be indexed as (111), (220) and (311) planes of the cubic phase of CdS (JCPDS #10-0454) (Hao et al. 2019). The bare RP cells did not show any obvious XRD peaks. As expected, the XRD pattern of RP/CdS hybrid cells also showed diffraction peaks attributed to CdS QDs (Fig. 2a). In addition, Raman analysis of the RP/CdS hybrid cells also showed a typical peak similar to that of pure CdS, while bare RP cells did not exhibit obvious peak (Fig. 2b). The distinct Raman peak at ~ 295 cm−1 could be identified as the longitudinal optical phonon resulting from the Cd–S bond vibration (Ye et al. 2020; Ma et al. 2020a, b), suggesting the presence of CdS on the bio-nano-hybrid cells. Furthermore, EDS analysis was also applied to analysis the elemental composition of the RP/CdS hybrid cells. It could be concluded that Cd element and S element coexisted with C, N, O, P elements in these hybrid cells, although the Cd element and S element were not prominent due to their low content (the CdS loading in the hybrid cell is 0.11 wt%) (Fig. 2c). These results confirmed that the CdS QDs was assembled onto the RP cell, demonstrating that the RP/CdS bio-nano-hybrid cells were successfully constructed.
Biomethanation performance of RP/CdS bio-nano-hybrid cells
In order to improve the biomethanation efficiency of the RP cell under visible light, the methane production of the bio-nano-hybrid cells was compared with that of the bare RP cells. The visible light was divided into three different groups with different wavelength ranges by using different optical filters (400–500, 500–600, 600–700 nm). Once the RP cells or RP/CdS bio-nano-hybrid cells were anaerobically incubated under light irradiation, methane gas gradually accumulated and reached the highest production at around 48 h (Fig. 3). It was found that bare RP cells delivered the highest methane production of about 17, 36, and 42 nmol/mg total protein under irradiation with light of 400–500, 500–600, and 600–700 nm, respectively (Fig. 3). Pure CdS QDs under the same conditions did not produce detectable methane (Fig. 3). Then, the methane production by the RP/CdS bio-nano-hybrid cells was determined. As shown in Fig. 3a, under the irradiation of light RP/CdS bio-nano-hybrid cells with the wavelength of 400–500 nm, the RP/CdS bio-nano-hybrid cells reached the highest methane production of 31 ± 0.6 nmol/mg total protein, while the bare RP cells only showed the highest production of 17.3 ± 0.6 nmol/mg total protein. Under light irradiation with the wavelength of 500–600 nm, there was no significant difference could be observed between bare RP cells and the RP/CdS bio-nano-hybrid cells (Fig. 3b). Under the light irradiation with the wavelength of 600–700 nm, only a very slight improvement (< 10%) was observed by the RP/CdS bio-nano-hybrid cells as compared to the bare RP cells (Fig. 3c). In accordance, CdS QDs prepared here had high adsorption at 400–500 nm, while only weak adsorption was observed over 500 nm (Additional file 1: Fig. S2). Thus, it is reasonable that the modification of RP cells with CdS QDs greatly improved the biomethanation efficiency at 400–500 nm, while only slight effect was observed over 500 nm.
As only sodium bicarbonate was presence as the carbon source and no nitrogen source was provided in the biomethanation medium of the RP/CdS bio-nano-hybrid cells, the hybrid cells solely used CO2 for methane production (Fixen et al. 2018). In addition, it was already proved that the genetically engineered RP strain converted the CO2 to methane with a single enzymatic step catalyzed by the mutated nitrogenase (Fixen et al. 2018; Ma et al. 2020a, b). The biomethanation (CO2-to-CH4) by mutated nitrogenase of RP strain with the photogenerated ATP (energy source) and electrons (reducing equivalent) harvested from the reducing agents (Additional file 1: Fig. S3) (Fixen et al. 2018; Zheng et al. 2018). Here, it could be proposed that the CdS QDs generated photoelectrons under visible light irradiation, which might be involved in the biomethanation process via two possible pathways (Additional file 1: Fig. S3). On the one hand, the photogenerated electrons from CdS QDs might directly inject into the nitrogenase with cellular electron transfer pathways (Chen et al. 2019), which might directly reinforce the reducing equivalent for improved methane production. On the other hand, the photogenerated electrons from CdS QDs might be injected into cell, which in turn induce the proton efflux and facilitate ATP generation (Fixen et al. 2018). The enhanced ATP generation might further provide extra energy source to improve the efficiency of biomethanation. However, the detailed mechanism is still unclear, which calls for further investigation.
Effects of different parameters on biomethanation of RP/CdS bio-nano-hybrid
With the aim of further improving the methane production of bio-nano-hybrid cells, the effects of different parameters (cell density, substrate concentration, L-Cys concentration, light intensity) on methane production of the RP/CdS bio-nano-hybrid cells were determined.
For biological product synthesis by microorganisms, cell density and substrate concentrations are essential parameters. It was found that, cell density significantly affected the methane production from the RP/CdS hybrid cells (Fig. 4a). Once the initial cell density of OD660 was over 0.5, the methane production decreased along with the increased cell density. The methane production could reach 30 ± 2.3 nmol/mg total protein with the OD660 of 0.1, which is comparable with that obtained under the OD660 of 0.5 (p > 0.1). The effect of substrate (sodium bicarbonate) concentration on the methane production was also evaluated. According to the results (Fig. 4b), no significant difference (p > 0.1) could be found among these tested concentrations. The result was reasonable as the minimum concentration of substrate used here was already saturated for methane production by this RP/CdS hybrid cells.
It is also well-known that light intensity and scavenger concentration have great impact on the bio-photocatalysis (Jiang et al. 2018). The hole/radical scavenger could accelerate the separation of photogenerated charge and remove the detrimental radical to avoid the inactivation of bacterial cell (Shen et al. 2020). Therefore, the concentration of L-Cys should be optimized. With the increase of L-Cys concentration from 2 to 14 mM, the methane production increased from 24 nmol/mg total protein to 38 ± 0.4 nmol/mg total protein (Fig. 4c). The light intensity should also be optimized as low intensity cannot provide enough energy for high catalytic activity while high intensity may result in photo-toxicity (Jiang et al. 2018). As shown in Fig. 4d, the bio-nano-hybrid cells could not produce any methane without light irradiation. With the increasing of light intensity (100–5000 lx), the methane production dramatically increased along with the light intensity from about 30 nmol/mg total protein to 168 ± 7 nmol/mg total protein. However, further increasing the light intensity to 7500 lx resulted in decreased methane production, which might be due to photo-toxicity. Among these parameters, it could be found that the light intensity was the most prominent factor that dramatically affected the methane production. Based on the effects of different parameters, the methane production from RP/CdS hybrid cells was tested under the optimum conditions (OD660 = 0.1, sodium bicarbonate = 2 mM, L-Cys = 14 mM, light intensity = 5000 lx) (Fig. 5). It was found that the methane production steadily increased upon inoculation and reached the highest production of 171 ± 10 nmol/mg total protein.